Dopaminergic neuron progenitor cell marker 187a5

ABSTRACT

An object of the present invention is to provide a probe, a primer, a primer set and an antibody for use in the detection or selection of a dopaminergic neuron progenitor cell. The present invention provides a probe, a primer and a primer set for use in the detection or selection of a mesencephalon dopaminergic neuron progenitor cell, and preferably a dopaminergic neuron proliferative progenitor cell, which can hybridize with a nucleotide sequence of a 187A5 gene, or a complementary sequence thereto, and an antibody for use in the detection or selection of a mesencephalon dopaminergic neuron progenitor cell, and preferably a dopaminergic neuron progenitor cell, which is capable of binding to a 187A5 protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of PCT/JP2007/058009, filedApr. 11, 2007, which claims priority to Japanese Patent Application No.2006-108786, filed Apr. 11, 2006. The contents of all of theaforementioned applications are herein incorporated by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to a 187A5 gene, which is a dopaminergicneuron progenitor cell marker. More particularly, the present inventionrelates to a means for detecting a dopaminergic neuron progenitor cell,a method for detecting the cell, and a kit for detecting the cell.

BACKGROUND ART

The dopamine system is a very important system involved in movementcontrol, hormone secretion control, affectivity control, and so forth,which are important in the mammalian brain. Therefore, abnormalities indopaminergic neurotransmission cause various disorders of the neuralsystem. For example, the Parkinson's disease is a neurodegenerativedisease of the extrapyramidal system which is caused by specificdegeneration of dopaminergic neurons in the mesencephalon substantianigra (HARRISON'S PRINCIPLES OF INTERNAL MEDICINE Vol. 2 23^(rd) ed.,Isselbacher et al. edited by McGraw-Hill Inc., NY (1994) pp. 2275-7).

As a method for treating the Parkinson's disease, a method of orallyadministering L-DOPA (3,4-dihydroxy-phenylalanine) has been mainlyadopted for compensating the decrease in the amount of the produceddopamine, but it is known that the duration of the effect is not good.

Accordingly, as a method for compensating the loss of dopaminergicneurons, recently, there has been attempted a therapeutic method oftransplanting a mesencephalon ventral region of a 6-9 week aborted fetuscontaining dopaminergic neuron precursors (U.S. Pat. No. 5,690,927;Spencer et al. (1992) N. Engl. J. Med. 327:1541-8; Freed et al. (1992)N. Engl. J. Med. 327:1549-55; Widner et al. (1992) N. Engl. J. Med.327:1556-63; Kordower et al. (1995) N. Engl. J. Med. 332:1118-24; Deferet al. (1996) Brain 119:41-50; and Lopez-Lozano et al. (1997) Transp.Proc. 29:977-80). However, at the present time, in addition to cellsupply and ethical issues (Rosenstain (1995) Exp. Neurol. 33:106; Turneret al. (1993) Neurosurg. 33:1031-7), various other problems have beenindicated, for example, risk of infectious contamination, immunologictransplant rejection (Lopez-Lozano et al. (1997) Transp. Proc. 29:977-80and Widner and Brudin (1988) Brain Res. Rev. 13:287-324), low survivalrate due to the fetus tissue's mainly dependence on lipid metabolismrather than glycolysis (Rosenstein (1995) Exp. Neurol. 33:106), and soforth.

As a method for solving the problem of the ethical issues or supplyshortage, for example, a method by using a cortex, a striatum, andmesencephalon cells, derived from a pig, and so forth have been proposed(for example, Japanese Patent Laid-Open Publication No. 10-508487, No.10-508488, and No. 10-509034). However, in this method, a complexprocedure for modifying an antigen on the cell surface (MHC class Iantigen) is required to suppress rejection. As a method for solving thetransplant rejection, for example, a method involving localimmunosuppression by simultaneously transplanting Sertoli cells has beenproposed (Japanese Patent Laid-Open Publication No. 11-509170 and No.11-501818; and Selawly and Cameron (1993) Cell Transplant 2:123-9). Itis possible that transplant cells are obtained from a relative whose MHCmatches, bone marrow of another person, a bone marrow bank, a cord bloodbank, and so forth. However, if patient's own cells can be used, theproblems of rejection can be solved without extra procedures andtrouble.

Accordingly, it has been expected that, instead of cells derived from anaborted fetus, a differentiation system of dopaminergic neurons in vitrofrom non-neural cells such as embryo-stem (ES) cell and bone marrowstromal cells are utilized as a transplant material. Actually, it isconfirmed that a dopaminergic neuron derived from ES cell is functionalfor transplantation into lesion striatum of a rat Parkinson's diseasemodel (Kim et al. (2002) Nature 418:50-56). It is thought that in thefuture, importance of regenerative medicine from ES cells or thepatient's own neural stem cells will increase.

On the other hand, in the treatment of damage of neural tissue,restructuring of brain function is required, and for forming appropriatelinkage with surrounding cells (network formation), not mature cells butprogenitor cells that can differentiate into neurons in vivo arerequired to be transplanted. However, in the transplantation of neuronprogenitor cells, in addition to the above-described problem regardingsupply, there is a problem that the progenitor cells can differentiateinto a nonuniform cell population. For example, in the treatment of theParkinson's disease, it is necessary that dopaminergic neurons areselectively transplanted among catecholamine-containing neurons. Beforenow, as transplant cells for use in the treatment of the Parkinson'sdisease, there has been proposed a striatum (Lindvall et al. (1989)Arch. Neurol. 46:615-31 and Widner et al. (1992) N. Engl. J. Med.327:1556-63), an immortalized cell line derived from human embryonicnerve (Japanese Patent Laid-Open Publication No. 8-509215, No.11-506930, and No. 2002-522070), a post-mitotic human neuron of NT2Zcells (Japanese Patent Laid-Open Publication No. 9-5050554), a neuronprimordial cell (Japanese Patent Laid-Open Publication No. 11-509729), acell transfected with an exogenous gene so as to produce catecholaminesuch as dopamine, a bone marrow stromal cell (Japanese Patent Laid-OpenPublication No. 2002-504503 and No. 2002-513545), an ES cell in which agene is modified (Kim et al. (2002) Nature 418:50-56), and so forth.However, none of these contain only dopaminergic neurons or cells todifferentiate into dopaminergic neurons.

As a method for selectively condensing or isolating dopaminergic neuronsfrom undifferentiated cell population, there has been proposed a methodof, introducing a reporter gene expressing a fluorescent protein undercontrol of promoter/enhancer of a gene such as tyrosine hydroxylase (TH)expressed in dopaminergic neurons into each cell of the cell population,isolating the cells emitting fluorescence, and thereby visualizing thealive dopaminergic neurons to condense, segregate or identify (JapanesePatent Laid-Open Publication No. 2002-51775). However, this methodrequires a complex step of introduction of an exogenous gene, andfurthermore, when used in gene treatment, the existence of the reportergene causes problem of toxicity and immunogenicity.

As described above, now, one of the largest problems in transplantationtreatment for the Parkinson's disease is that the either dopaminergicneuron progenitor cells derived from the mesencephalon ventral region ofaborted fetus or induced to differentiate are a mixture of variouscells. It is desirable that only a desired cell species is isolated andused, considering safety in neural network formation. Furthermore,considering survival or ability for correctly forming a network in abrain in which the cells are transplanted, it can be said that it isdesirable from the treatment effect that earlier proliferativeprogenitor cells are isolated and transplanted.

Before now, as a gene that is expressed in the dopaminergic neuronproliferative progenitor cells, Lrp4 (WO 2004/065599) has been reported.Additionally, some markers of dopaminergic neuron progenitor cells havebeen reported (WO 2004/038018 and WO 2004/052190).

SUMMARY OF THE INVENTION

In order to isolate a gene selectively expressed in dopaminergic neuronprogenitor cells, and preferably dopaminergic neuron proliferativeprogenitor cells, the present inventors have separated cells positivefor an Lrp4 protein, a dopaminergic neuron proliferative progenitor cellmarker gene, from the mesencephalon and metencephalon ventral regions ofa 13.5-day rat embryo, and have searched a gene specific for theLrp4-positive cells in the mesencephalon by a subtraction (N-RDA)method. The present inventors have consequently found a gene selectivelyexpressed in dopaminergic neuron proliferative progenitor cells (187A5gene (hereinafter, occasionally referred to as “187A5”)) (Example 2).The present invention is based on this finding.

An object of the present invention is to provide a means for detecting adopaminergic neuron progenitor cell (preferably, a dopaminergic neuronproliferative progenitor cell), a method for detecting a dopaminergicneuron progenitor cell (preferably, a dopaminergic neuron proliferativeprogenitor cell), and a kit for detecting a dopaminergic neuronprogenitor cell (preferably, a dopaminergic neuron proliferativeprogenitor cell).

Further, an object of the present invention is to provide a method forscreening for an effective substance for inducing differentiation into adopaminergic neuron progenitor cell (preferably, a dopaminergic neuronproliferative progenitor cell).

Furthermore, an object of the present invention is to provide a methodfor producing a dopaminergic neuron progenitor cell (preferably, adopaminergic neuron proliferative progenitor cell) for use in thetreatment of the Parkinson's disease.

The present invention provides a polynucleotide selected from thefollowing (i), (ii), (iii) and (iv) (hereinafter, occasionally referredto as a “187A5 gene”):

(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;(ii) a polynucleotide encoding a protein which consists of an amino acidsequence encoded by a nucleotide sequence of SEQ ID NO: 1 in which oneor more nucleotides are inserted, substituted and/or deleted, and/or oneor more nucleotides are added to one or both of ends, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2;(iii) a polynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; and(iv) a polynucleotide which has 70% or more identity with apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a protein selected from thefollowing (v), (vi), (vii) and (viii) (hereinafter, occasionallyreferred to as a “187A5 protein”):

(v) a protein comprising the amino acid sequence of SEQ ID NO: 2;(vi) a protein which consists of an amino acid sequence of SEQ ID NO: 2in which one or more amino acids are inserted, substituted and/ordeleted, and/or one or more amino acids are added to one or both ofends, and which is functionally equivalent to a protein consisting ofthe amino acid sequence of SEQ ID NO: 2;(vii) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide which encodes the aminoacid sequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2.

The present invention provides a probe or primer for use in thedetection or selection of a dopaminergic neuron progenitor cell(preferably, a dopaminergic neuron proliferative progenitor cell), whichcan hybridize to a nucleotide sequence of a 187A5 gene, or acomplementary sequence thereto (hereinafter, occasionally referred to asa “probe according to the present invention” and a “primer according tothe present invention”, respectively).

The present invention provides an antibody for use in the detection orselection of a dopaminergic neuron progenitor cell (preferably, adopaminergic neuron proliferative progenitor cell), which is capable ofbinding to a 187A5 protein (hereinafter, occasionally referred to as an“antibody according to the present invention”).

The present invention provides a method for detecting or selecting adopaminergic neuron progenitor cell (preferably, a dopaminergic neuronproliferative progenitor cell), comprising the step of detectingexpression of a 187A5 gene, or a 187A5 protein (hereinafter,occasionally referred to as a “detection method according to the presentinvention”).

The present invention provides a kit for detecting or selecting adopaminergic neuron progenitor cell (preferably, a dopaminergic neuronproliferative progenitor cell), comprising at least a probe according tothe present invention, a primer according to the present invention, aprimer set according to the present invention, or an antibody accordingto the present invention (hereinafter, occasionally referred to as a“detection kit according to the present invention”).

The present invention provides an agent for detecting or selecting adopaminergic neuron progenitor cell (preferably, a dopaminergic neuronproliferative progenitor cell), comprising at least a probe according tothe present invention, a primer according to the present invention, aprimer set according to the present invention, or an antibody accordingto the present invention (hereinafter, occasionally referred to as an“agent for detection according to the present invention”).

The present invention provides a method for screening for an effectivesubstance for inducing differentiation into a dopaminergic neuronprogenitor cell (preferably, a dopaminergic neuron proliferativeprogenitor cell), comprising the step of detecting expression of a 187A5gene, or a 187A5 protein.

The present invention provides a method for producing a dopaminergicneuron progenitor cell (preferably, a dopaminergic neuron proliferativeprogenitor cell) for use in the treatment of the Parkinson's disease.

The probe according to the present invention, the primer according tothe present invention, the primer set according to the present inventionand the antibody according to the present invention can be used asmarkers specific for dopaminergic neuron progenitor cells, andpreferably dopaminergic neuron proliferative progenitor cells in themesencephalon. Accordingly, the present invention is extremely useful ina purity test of a transplant material and development of a method forinducing differentiation into a dopaminergic neuron progenitor cell, andpreferably a dopaminergic neuron proliferative progenitor cell in vitro,or the like, and largely contributes to the promotion of practicalapplication of regenerative medicine. Moreover, the protein according tothe present invention is not merely expressed but has a region expressedin the extracellular space. Accordingly, the extracellular region of theprotein according to the present invention can be used as an index fordetecting live cells with reliability and for separating and obtainingthe cells. Therefore, the present invention is expected to largelycontribute to the practical application of regenerative medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an expression period of dopaminergic neuron-related markergenes.

FIG. 2 shows the results of analyzing, by an immunostaining method,protein expressions of Lrp4 and TH in the mesencephalon andmetencephalon of a 14.5-day rat embryo.

FIG. 3 shows the results of analyzing, by a RT-PCR method, mRNAexpressions of 187A5, Lmx1a and Lrp4 in mesencephalon and metencephalonLrp4-positive cells.

FIG. 4 shows the results of analyzing, by in situ hybridization, mRNAexpressions of 187A5 and Lrp4 in the mesencephalon and metencephalon ofa 12.5-day mouse embryo.

FIG. 5 shows the results of analyzing, by a RT-PCR method, mRNAexpressions of 187A5, Lmx1a and Lrp4 in dopaminergic neuronproliferative progenitor cells induced to differentiate from ES cells byan SDIA method.

FIG. 6 shows the results of analyzing, by a RT-PCR method, mRNAexpressions of 187A5, Lmx1a and Lrp4 in dopaminergic neuron progenitorcells induced to differentiate from ES cells by a 5-stage method.

FIG. 7 shows mouse 187A5 and 187A5-SEAP.

FIG. 8 shows the results of analyzing signal sequence activity of 187A5.

FIG. 9 shows the results of analyzing expression of a 187A5 protein onthe cell surface by a biotinylation method of cell surface proteins.

FIG. 10 shows the results of investigating expression of a 187A5 proteinon the cell surface by FACS analysis. In the drawing, a boxed arearepresents 187A5-expressing cells.

FIG. 11 shows the results of analyzing, by an immunostaining method,expression of a 187A5 protein in the mesencephalon and metencephalonventral regions of an 11.5-day mouse embryo.

FIG. 12 shows the results of investigating, by FACS analysis,expressions of 187A5 and Lrp4 proteins in the mesencephalon andmetencephalon ventral regions of a 12.5-day mouse embryo.

FIG. 13 shows the results of investigating, by FACS analysis,expressions of 187A5 and Lrp4 proteins in dopaminergic neuronproliferative progenitor cells induced to differentiate from ES cells byan SDIA method.

FIG. 14 shows the results of separating 187A5/Lrp4-copositive cellsinduced to differentiate from ES cells by an SDIA method, and culturing.

FIG. 15 schematically shows the structure of a DNA construct that can beused for selecting dopaminergic neuron progenitor cells.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be explained in detail. Thefollowing description is an example for explaining the presentinvention, and the present invention is not limited to the embodimentsto be described. All technical terms, scientific terms and terminologiesused in the present specification have the same meanings as those thatare generally understood by those ordinary skilled in the art in thetechnical fields to which the present invention belongs, and are usedmerely for the purpose of explaining a specific embodiment but are notintended to make limitation. The present invention can be carried out invarious embodiments as long as not departing from the spirit thereof.All the prior art documents, published publications, patent publicationsand other patent documents cited in the present specification areincorporated into the present specification as references, and can beused for carrying out the present invention.

[Dopaminergic Neuron Progenitor Cell]

The “dopaminergic neuron progenitor cell”, which is an object to bedetected or selected in the present invention, means prematuredopaminergic neuron cells.

The “dopaminergic neuron proliferative progenitor cell”, which is alsoan object to be detected or selected in the present invention, meansdopaminergic neuron progenitor cells before arrest of mitotic division.

Dopaminergic neurons differentiate from neuroepithelial cells, throughthe differentiation stages of proliferative progenitor cells andpostmitotic precursor cells, into mature dopaminergic neurons. Thedopaminergic neuron progenitor cells are progenitor cells in thedopaminergic neurons. Among them, the dopaminergic neuron proliferativeprogenitor cell is the earliest progenitor cell in the dopaminergicneurons, and therefore, high survival rate and high ability of networkformation in the brain to which the cell is transplanted can beexpected. Therefore, the dopaminergic neuron progenitor cell,particularly, the dopaminergic neuron proliferative progenitor cell isuseful for the transplantation treatment of diseases caused by decreasein dopamine due to degeneration of the dopaminergic neurons, such as theParkinson's disease.

The cells selected by using the probe according to the presentinvention, the primer according to the present invention, the primer setaccording to the present invention or the antibody according to thepresent invention as an index are dopaminergic neuron progenitor cells,and therefore, are preferable for the transplantation treatment ofneurodegenerative diseases such as the Parkinson's disease in theaspects of safety, survival rate and network formation ability, comparedto a conventional mixed cell population or dopaminergic neuronprogenitor cells in which an exogenous gene is introduced. Particularly,when the cells detected or selected by using the probe according to thepresent invention, the primer according to the present invention, theprimer set according to the present invention or the antibody accordingto the present invention are dopaminergic neuron progenitor cells beforearrest of mitotic division, namely, dopaminergic neuron progenitor cellsin proliferation, the cells have the possibility of differentiating tomature in the most appropriate place in the brain, and also, thedopaminergic neuron progenitor cells have the possibility ofproliferating in vivo. Therefore, a longer effect of the treatment canbe expected. Therefore, it can be said that the present invention pavesthe way to the practical application of the effective transplantationtreatment of neurodegenerative diseases such as the Parkinson's disease.

[187A5 Gene and Protein]

In the present invention, the “187A5 gene” means those encoding a 187A5protein and includes not only cDNA but also genomic DNA. It alsoincludes RNA corresponding thereto.

In the present invention, the “187A5 gene”, which is an index for theexistence of dopaminergic neuron progenitor cells, has been registeredin database as a functionally unknown sequence in mice. However, inhumans, rats, bovines, dogs, chimpanzees, and so forth, only predictedsequences of 187A5 genes have been obtained. GenBank Accession Numbersdisclosing the respective sequences are as follows.

Human: XM_(—)044062 (SEQ ID NO: 3 (base sequence), SEQ ID NO: 4 (aminoacid sequence), hereinafter, representation will be in the same order),AK126715 (SEQ ID NO: 5, SEQ ID NO: 6), HSM803256 (SEQ ID NO: 7, SEQ IDNO: 8), HSM803467 (SEQ ID NO: 9, SEQ ID NO: 10)Mouse: AK028289 (SEQ ID NO: 11, SEQ ID NO: 12), AK157823 (SEQ ID NO: 13(base sequence)), AK028541 (SEQ ID NO: 14, SEQ ID NO: 15), AK035053 (SEQID NO: 16, SEQ ID NO: 17), XM_(—)485684 (SEQ ID NO: 18, SEQ ID NO: 19),AK163356 (SEQ ID NO: 20 (base sequence))

Rat: XM_(—)344107 (SEQ ID NO: 21, SEQ ID NO: 22) Bovine: XM_(—)590147(SEQ ID NO: 23, SEQ ID NO: 24) Dog: XM_(—)543360 (SEQ ID NO: 25, SEQ IDNO: 26) Chimpanzee: XM_(—)522557 (SEQ ID NO: 27, SEQ ID NO: 28)

In the present invention, a cDNA sequence (SEQ ID NO: 1) of a human187A5 gene as a gene selectively expressed in dopaminergic neuronprogenitor cells (preferably, dopaminergic neuron proliferativeprogenitor cells) in a mesencephalon site as well as an amino acidsequence (SEQ ID NO: 2) of a human 187A5 protein (polypeptide) as aprotein (polypeptide) selectively expressed in dopaminergic neuronprogenitor cells (preferably, dopaminergic neuron proliferativeprogenitor cells) were determined.

Those skilled in the art can specify a nucleotide sequence of a 187A5gene or an amino acid sequence of a 187A5 protein inherent in variousanimals based on the nucleotide sequence of the 187A5 gene of SEQ ID NO:1 and the amino acid sequence of the 187A5 protein of SEQ ID NO: 2. Forexample, by homology search based on the human or mouse 187A5 gene or187A5 protein, a 187A5 gene or a 187A5 protein of the animal can besearched and identified. In the homology search, BLAST to be describedlater or the like can be used. Therefore, in the present invention, the“187A5 gene” and the “187A5 protein” used is meant to include, inaddition to a human-derived 187A5 gene or a human-derived 187A5 protein,a 187A5 gene or a 187A5 protein inherent in various animals (preferably,mammals).

The 187A5 gene includes:

a polynucleotide encoding a human 187A5 protein comprising the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8or SEQ ID NO: 10;

a polynucleotide encoding a mouse 187A5 protein comprising the aminoacid sequence of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ IDNO: 19;

a polynucleotide encoding a rat 187A5 protein comprising the amino acidsequence of SEQ ID NO: 22;

a polynucleotide encoding a bovine 187A5 protein comprising the aminoacid sequence of SEQ ID NO: 24;

a polynucleotide encoding a dog 187A5 protein comprising the amino acidsequence of SEQ ID NO: 26; and

a polynucleotide encoding a chimpanzee 187A5 protein comprising theamino acid sequence of SEQ ID NO: 28.

Moreover, the 187A5 gene includes:

a polynucleotide comprising the human 187A5 gene nucleotide sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9;

a polynucleotide comprising the mouse 187A5 gene nucleotide sequence ofSEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18 or SEQ ID NO: 20;

a polynucleotide comprising the rat 187A5 gene nucleotide sequence ofSEQ ID NO: 21;

a polynucleotide comprising the bovine 187A5 gene nucleotide sequence ofSEQ ID NO: 23; a polynucleotide comprising the dog 187A5 gene nucleotidesequence of SEQ ID NO: 25; and

a polynucleotide comprising the chimpanzee 187A5 gene nucleotidesequence of SEQ ID NO: 27.

The 187A5 gene includes a polynucleotide selected from the following(i′), (ii′), (iii′) and (iv′):

(i′) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27;(ii′) a polynucleotide encoding a protein which consists of an aminoacid sequence encoded by a nucleotide sequence of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 in which one ormore nucleotides are inserted, substituted and/or deleted, and/or one ormore nucleotides are added to one or both of ends, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2;(iii′) a polynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, andwhich encodes a protein functionally equivalent to a protein consistingof the amino acid sequence of SEQ ID NO: 2; and(iv′) a polynucleotide which has 70% or more identity with apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, andwhich encodes a protein functionally equivalent to a protein consistingof the amino acid sequence of SEQ ID NO: 2.

Moreover, the 187A5 gene includes a polynucleotide encoding a proteinselected from the following (v′), (vi′), (vii′) and (viii′):

(v′) a protein comprising the amino acid sequence of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26 or SEQ ID NO: 28;(vi′) a protein which consists of an amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ IDNO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 in which one or more amino acidsare inserted, substituted and/or deleted, and/or one or more amino acidsare added to one or both of ends, and which is functionally equivalentto a protein consisting of the amino acid sequence of SEQ ID NO: 2;(vii′) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide which encodes the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, andwhich is functionally equivalent to a protein consisting of the aminoacid sequence of SEQ ID NO: 2; and(viii′) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26 or SEQ ID NO: 28, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2.

According to the present invention, preferably, there is provided apolynucleotide selected from the following (i″), (ii″), (iii″) and(iv″):

(i″) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1;(ii″) a polynucleotide encoding a protein which consists of an aminoacid sequence encoded by a nucleotide sequence of SEQ ID NO: 1 in whichone or more nucleotides are inserted, substituted and/or deleted, and/orone or more nucleotides are added to one or both of ends, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2;(iii″) a polynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; and(iv″) a polynucleotide which has 70% or more identity with apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2.

According to the present invention, preferably, there is also provided apolynucleotide encoding a protein selected from the following (v″),(vi″), (vii″) and (viii″):

(v″) a protein comprising the amino acid sequence of SEQ ID NO: 2;(vi″) a protein which consists of an amino acid sequence of SEQ ID NO: 2in which one or more amino acids are inserted, substituted and/ordeleted, and/or one or more amino acids are added to one or both ofends, and which is functionally equivalent to a protein consisting ofthe amino acid sequence of SEQ ID NO: 2;(vii″) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide which encodes the aminoacid sequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii″) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2.

According to the present invention, more preferably, there is provided ahuman-derived polynucleotide selected from the following (i′″), (ii′″),(iii′″) and (iv′″):

(i′″) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1;(ii′″) a polynucleotide encoding a protein which consists of an aminoacid sequence encoded by a nucleotide sequence of SEQ ID NO: 1 in whichone or more nucleotides are inserted, substituted and/or deleted, and/orone or more nucleotides are added to one or both of ends, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2;(iii′″) a polynucleotide which hybridizes under stringent conditions toa polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; and(iv′″) a polynucleotide which has 95% or more identity with apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2.

According to the present invention, more preferably, there is alsoprovided a polynucleotide encoding a human-derived protein selected fromthe following (v′″), (vi′″), (vii′″) and (viii′″):

(v′″) a protein comprising the amino acid sequence of SEQ ID NO: 2;(vi′″) a protein which consists of an amino acid sequence of SEQ ID NO:2 in which one or more amino acids are inserted, substituted and/ordeleted, and/or one or more amino acids are added to one or both ofends, and which is functionally equivalent to a protein consisting ofthe amino acid sequence of SEQ ID NO: 2;(vii′″) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide which encodes the aminoacid sequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii′″) a protein which consists of an amino acid sequence having 95%or more identity with the amino acid sequence of SEQ ID NO: 2, and whichis functionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2.

According to the present invention, further preferably, there isprovided a human-derived polynucleotide comprising the nucleotidesequence of SEQ ID NO: 1.

According to the present invention, further preferably, there is alsoprovided a polynucleotide encoding a human-derived protein comprisingthe amino acid sequence of SEQ ID NO: 2.

The 187A5 protein (polypeptide) includes:

a human 187A5 protein comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

a mouse 187A5 protein comprising the amino acid sequence of SEQ ID NO:12, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19,

a rat 187A5 protein comprising the amino acid sequence of SEQ ID NO: 22;

a bovine 187A5 protein comprising the amino acid sequence of SEQ ID NO:24;

a dog 187A5 protein comprising the amino acid sequence of SEQ ID NO: 26;and

a chimpanzee 187A5 protein comprising the amino acid sequence of SEQ IDNO: 28.

Moreover, the 187A5 protein (polypeptide) includes:

a protein which is encoded by a nucleotide sequence comprising the human187A5 gene nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 7 or SEQ ID NO: 9,

a protein which is encoded by a nucleotide sequence comprising the mouse187A5 gene nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

a protein which is encoded by a nucleotide sequence comprising the rat187A5 gene nucleotide sequence of SEQ ID NO: 21;

a protein which is encoded by a nucleotide sequence comprising thebovine 187A5 gene nucleotide sequence of SEQ ID NO: 23;

a protein which is encoded by a nucleotide sequence comprising the dog187A5 gene nucleotide sequence of SEQ ID NO: 25; and

a protein which is encoded by a nucleotide sequence comprising thechimpanzee 187A5 gene nucleotide sequence of SEQ ID NO: 27.

The 187A5 protein (polypeptide) includes a protein selected from thefollowing (i′), (ii′), (iii′) and (iv′):

(i′) a protein which is encoded by the nucleotide sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27;(ii′) a protein which consists of an amino acid sequence encoded by anucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 25 or SEQ ID NO: 27 in which one or more nucleotides areinserted, substituted and/or deleted, and/or one or more nucleotides areadded to one or both of ends, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2;(iii′) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 25 or SEQ ID NO: 27, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(iv′) a protein which is encoded by a polynucleotide which has 70% ormore identity with a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25 or SEQ ID NO: 27, and which is functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2.

Moreover, the 187A5 protein (polypeptide) includes a protein selectedfrom the following (v′), (vi′), (vii′) and (viii′):

(v′) a protein comprising the amino acid sequence of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26 or SEQ ID NO: 28;(vi′) a protein which consists of an amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ IDNO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 in which one or more amino acidsare inserted, substituted and/or deleted, and/or one or more amino acidsare added to one or both of ends, and which is functionally equivalentto a protein consisting of the amino acid sequence of SEQ ID NO: 2;(vii′) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide which encodes the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, andwhich is functionally equivalent to a protein consisting of the aminoacid sequence of SEQ ID NO: 2; and(viii′) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26 or SEQ ID NO: 28, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2.

According to the present invention, preferably, there is provided aprotein (polypeptide) selected from the following (i″), (ii″), (iii″)and (iv″):

(i″) a protein which is encoded by the nucleotide sequence of SEQ ID NO:1;(ii″) a protein which consists of an amino acid sequence encoded by anucleotide sequence of SEQ ID NO: 1 in which one or more nucleotides areinserted, substituted and/or deleted, and/or one or more nucleotides areadded to one or both of ends, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2;(iii″) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 1, and which is functionallyequivalent to a protein consisting of the amino acid sequence of SEQ IDNO: 2; and(iv″) a protein which is encoded by a polynucleotide which has 70% ormore identity with a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 1, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2.

According to the present invention, preferably, there is also provided aprotein (polypeptide) selected from the following (v″), (vi″), (vii″)and (viii″):

(v″) a protein comprising the amino acid sequence of SEQ ID NO: 2;(vi″) a protein which consists of an amino acid sequence of SEQ ID NO: 2in which one or more amino acids are inserted, substituted and/ordeleted, and/or one or more amino acids are added to one or both ofends, and which is functionally equivalent to a protein consisting ofthe amino acid sequence of SEQ ID NO: 2;(vii″) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide which encodes the aminoacid sequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii″) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2.

According to the present invention, more preferably, there is provided ahuman-derived protein (polypeptide) selected from the following (i′″),(ii′″), (iii′″) and (iv′″):

(i′″) a protein which is encoded by the nucleotide sequence of SEQ IDNO: 1;(ii′″) a protein which consists of an amino acid sequence encoded by anucleotide sequence of SEQ ID NO: 1 in which one or more nucleotides areinserted, substituted and/or deleted, and/or one or more nucleotides areadded to one or both of ends, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2;(iii′″) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 1, and which is functionallyequivalent to a protein consisting of the amino acid sequence of SEQ IDNO: 2; and(iv′″) a protein which is encoded by a polynucleotide which has 95% ormore identity with a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 1, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2.

According to the present invention, more preferably, there is alsoprovided a human-derived protein (polypeptide) selected from thefollowing (v′″), (vi′″), (vii′″) and (viii′″):

(v′″) a protein comprising the amino acid sequence of SEQ ID NO: 2;(vi′″) a protein which consists of an amino acid sequence of SEQ ID NO:2 in which one or more amino acids are inserted, substituted and/ordeleted, and/or one or more amino acids are added to one or both ofends, and which is functionally equivalent to a protein consisting ofthe amino acid sequence of SEQ ID NO: 2;(vii′″) a protein which is encoded by a polynucleotide which hybridizesunder stringent conditions to a polynucleotide which encodes the aminoacid sequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii′″) a protein which consists of an amino acid sequence having 95%or more identity with the amino acid sequence of SEQ ID NO: 2, and whichis functionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2.

According to the present invention, further preferably, there isprovided a human-derived protein encoded by the nucleotide sequence ofSEQ ID NO: 1.

According to the present invention, further preferably, there is alsoprovided a human-derived protein comprising the amino acid sequence ofSEQ ID NO: 2.

In the present specification, “a polynucleotide in which one or morenucleotides are inserted, substituted and/or deleted, and/or one or morenucleotides are added to one or both of ends” or “an amino acid sequencein which one or more amino acids are inserted, substituted and/ordeleted, and/or one or more amino acids are added to one or both ofends” means that the modification is performed by a well-known technicalmethod such as site-directed mutagenesis or by substitution of aplurality of nucleotides or amino acids to an extent of being naturallygenerated, or the like. In the case of the polynucleotide, the term ismeant to include single nucleotide polymorphisms (SNPs). The number ofnucleotide or amino acid modifications can be insertion, substitution,deletion, and/or addition to one or both of ends, of, for example, 1 to30, preferably 1 to 20, more preferably 1 to 10, further preferably oneto several (for example, 9 or less), particularly preferably 1 to 4, andmost preferably 1 or 2 nucleotides or amino acids.

The modified nucleotide sequence can be preferably a nucleotide sequenceof SEQ ID NO: 1 having one or more (for example, one or several, or 1,2, 3 or 4) mutations without affecting the functions of a proteinconsisting of the amino acid sequence of SEQ ID NO: 2.

The modified amino acid sequence can be preferably an amino acidsequence of SEQ ID NO: 2 having one or more (for example, one orseveral, or 1, 2, 3 or 4) conservative substitutions.

The number of insertion, substitution, deletion or addition introducedinto the nucleotide sequence in (ii), (ii′), (ii″) or (ii′″) can bepreferably one or several (for example, 9 or less), more preferably 1 to6, particularly preferably 1 to 4, most preferably 1 or 2.

The number of insertion, substitution, deletion or addition introducedinto the amino acid sequence in (vi), (vi′), (vi″) or (vi′″) can bepreferably one or several (for example, 9 or less), more preferably 1 to6, particularly preferably 1 to 4, most preferably 1 or 2.

In the present specification, the “conservative substitutions” mean thatone or more amino acid residues are substituted with other chemicallyanalogous amino acid residues so as not to substantially change proteinfunctions. For example, the case that a certain hydrophobic residue issubstituted with another hydrophobic residue and the case that a certainpolar residue is substituted with another polar residue having the samecharge can be exemplified. Functionally analogous amino acids which canbe substituted in such a manner are known in the technical field, withrespect to every amino acid. To give specific examples, non-polar(hydrophobic) amino acids include alanine, valine, isoleucine, leucine,proline, tryptophan, phenylalanine and methionine. Polar (neutral) aminoacids include glycine, serine, threonine, tyrosine, glutamine,asparagine and cysteine. Positively charged (basic) amino acids includearginine, histidine and lysine. Negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

The modified nucleotide sequence includes a nucleotide sequence havingsubstitution of guanine to adenine at nucleotide 512 of SEQ ID NO: 1,substitution of guanine to adenine at nucleotide 844 of SEQ ID NO: 1,substitution of guanine to adenine at nucleotide 1360 of SEQ ID NO: 1,substitution of adenine to guanine at nucleotide 2458 of SEQ ID NO: 1 orsubstitution of adenine to guanine at nucleotide 2991 of SEQ ID NO: 1.The modified nucleotide sequence may have all or some of thesesubstitutions in combination.

The modified amino acid sequence includes an amino acid sequence havingsubstitution of arginine to histidine at amino acid 161 of SEQ ID NO: 2,substitution of valine to isoleucine at amino acid 272 of SEQ ID NO: 2,substitution of valine to isoleucine at amino acid 444 of SEQ ID NO: 2or substitution of arginine to glycine at amino acid 810 of SEQ ID NO:2. The modified amino acid sequence may have all or some of thesesubstitutions in combination.

In the present specification, “hybridize under stringent conditions”means hybridization to a target polynucleotide under stringentconditions. Specifically, there can be exemplified a polynucleotidehaving at least 70% or more, preferably 80% or more, more preferably 85%or more, further preferably 90% or more, further more preferably 95% ormore, particularly preferably 98% or more, and most preferably 99% ormore identity, with the target nucleotide sequence when calculation isperformed using a parameter of default (initial setting) with homologysearch software such as FASTA, BLAST or Smith-Waterman (Meth. Enzym.,164, 765 (1988)). Moreover, the “stringent conditions” can be performedaccording to a method of performing reaction in a hybridization bufferthat can be generally used by those skilled in the art so that thetemperature is 40 to 70° C., and preferably 60 to 65° C., and performingrinsing in a rinse solution whose salt concentration is 15 to 300mmol/L, and preferably 15 to 60 mmol/L. The temperature and the saltconcentration can be appropriately adjusted according to a length of theprobe to be used. Furthermore, the condition when the hybridizednucleotide is rinsed can be 0.2 or 2×SSC, 0.1% SDS, and a temperature of20 to 68° C. As to control of stringent (high stringency) or mild (lowstringency) conditions, the difference can be provided by a saltconcentration or a temperature in rinsing. When the difference of thehybridization is provided by a salt concentration, a stringent washbuffer (high stringency wash buffer) of 0.2×SSC, 0.1% SDS, or a mildwash buffer (low stringency wash buffer) of 2×SSC, 0.1% SDS can be used.Alternatively, when the difference of the hybridization is provided by atemperature, the temperature is 68° C. in the stringent case, 42° C. inthe case of moderate stringency, and room temperature (20 to 25° C.) inthe mild case, and every case thereof may be performed under 0.2×SSC,0.1% SDS.

In general, prehybridization is performed under the same conditions asthe hybridization. However, hybridization and preliminary rinsing arenot limited to be performed under the same conditions.

The hybridization can be performed according to a known method.Moreover, in the case of using a commercially available library, thehybridization can be performed according to the method described in theappended instruction for use.

In the present specification, the “identity” (occasionally referred toas homology) with respect to amino acid sequences means the degree ofidentity of the amino acid residues of the respective sequences betweenthe sequences to be compared. In this case, existence of a gap andproperties of the amino acids are considered (Wilbur, Natl. Acad. Sci.U.S.A. 80: 726-730 (1983)). For calculation of the homology,commercially available software BLAST (Altschul: J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron: Methods in Enzymology 183: 63-69(1990)), or the like can be used.

The “identity” may be a value calculated by using a homology searchprogram known by those skilled in the art and can be calculated, forexample, by using a parameter of default (initial setting) in thehomology algorithm BLAST (Basic local alignment search tool)http://www.ncbi.nlm.nih.gov/BLAST/in NCBI (National Center forBiotechnology Information).

The nucleotide sequence having at least 70% or more identity with thenucleotide sequence of SEQ ID NO: 1 can be a nucleotide sequence havingpreferably 80% or more, more preferably 90% or more, further preferably90% or more, further more preferably 95% or more, particularlypreferably 98% or more, and most preferably 99% or more identity.

The amino acid sequence having at least 70% or more identity with theamino acid sequence of SEQ ID NO: 2 can be an amino acid sequence havingpreferably 80% or more, more preferably 90% or more, further preferably90% or more, further more preferably 95% or more, particularlypreferably 98% or more, and most preferably 99% or more identity.

In the present invention, if the amino acid sequence of SEQ ID NO: 2 isgiven, a nucleotide sequence encoding it can be easily determined, andthereby, various nucleotide sequences encoding the amino acid sequenceof SEQ ID NO: 2 can be selected. Thus, a polynucleotide encoding aprotein consisting of the amino acid sequence of SEQ ID NO: 2 means notonly a part or all of a cDNA sequence of SEQ ID NO: 1 but also a cDNAsequence encoding the same amino acids, which has a codon having adegeneracy relationship therewith as a cDNA sequence. Furthermore, thepolynucleotide encoding a protein consisting of the amino acid sequenceof SEQ ID NO: 2 means even a genomic DNA sequence also containingintrons or noncoding regions. In the present invention, it furtherincludes an RNA sequence corresponding thereto.

In the present specification, whether or not to be “functionallyequivalent to a protein consisting of the amino acid sequence of SEQ IDNO: 2” can be determined by evaluating a biological phenomenon orfunctions associated with the expression of the 187A5 gene. For example,it can be determined by evaluating whether or not to be selectivelyexpressed in dopaminergic neuron progenitor cells, and preferablydopaminergic neuron proliferative progenitor cells in the mesencephalon.

The present invention provides a protein comprising a polypeptideconsisting of at least 5 amino acid residues (preferably, at least 6amino acid residues) or all of an amino acid sequence of amino acids248-397 or 792-877 of SEQ ID NO: 2. This protein corresponds to a highdiscrimination part in the amino acid sequence of the 187A5 protein, andtherefore, can be used as an antigen against an antibody that candiscriminate the 187A5 protein with higher accuracy.

The 187A5 protein is a type I single transmembrane protein that isexpressed on the cell surface in a direction wherein the N-terminal sidethereof can be located in the extracellular space. Thus, by flowcytometry using an antibody capable of binding to the protein, livecells in which the protein is expressed can be separated.

The present invention provides a protein comprising a polypeptideconsisting of at least 5 amino acid residues (preferably, at least 6amino acid residues) or all of an amino acid sequence of amino acids 28to 927 of SEQ ID NO: 2, amino acids 16 to 1267 of SEQ ID NO: 4, aminoacids 1 to 550 of SEQ ID NO: 6, amino acids 1 to 542 of SEQ ID NO: 8,amino acids 1 to 418 of SEQ ID NO: 10, amino acids 76 to 964 of SEQ IDNO: 12, amino acids 40 to 928 of SEQ ID NO: 15, amino acids 1 to 540 ofSEQ ID NO: 17, amino acids 40 to 1106 of SEQ ID NO: 19, amino acids 24to 1524 of SEQ ID NO: 22, amino acids 43 to 1018 of SEQ ID NO: 24, aminoacids 43 to 908 of SEQ ID NO: 26 or amino acids 1 to 866 of SEQ ID NO:28. This protein corresponds to the extracellular region in the aminoacid sequence of the 187A5 protein, and therefore, can be used as anantigen for preparing an antibody that can detect live cells as anobject to be detected.

The present invention provides use of the protein according to thepresent invention as an index for detecting or selecting a dopaminergicneuron progenitor cell, and preferably a dopaminergic neuronproliferative progenitor cell.

[Probe, Primer and Primer Set]

The probe or primer according to the present invention for use in thedetection or selection of a dopaminergic neuron progenitor cell, andpreferably a dopaminergic neuron proliferative progenitor cell canspecifically hybridize to a 187A5 gene. According to Example 2, in a12.5-day mouse embryo which is in the period of generating dopaminergicneurons, mRNA of 187A5 is selectively expressed in the mesencephalonmost ventral ventricular zone (ventricular zone; VZ) and themesencephalon most dorsal roof plate zone in which Lrp4-positivedopaminergic neuron progenitor cells exist, but is not expressed inmetencephalon floor plate cells positive for Lrp4. Therefore, it becamerevealed that mRNA of 187A5 is selectively expressed in dopaminergicneuron proliferative progenitor cells. Accordingly, the expression ofthe 187A5 gene is useful as an index for dopaminergic neuron progenitorcells. Therefore, the probe, the primer and the primer set according tothe present invention can be used as a marker for detecting dopaminergicneuron progenitor cells, and preferably dopaminergic neuronproliferative progenitor cells.

The probe and the primer according to the present invention can be usedfor detecting expression of a 187A5 gene, and corresponds to a polymerconsisting of a plurality of bases or base pairs such asdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It is known thatdouble-strand cDNA can also be used in tissue in situ hybridization, andsuch double-strand cDNA is also included in the probe and the primeraccording to the present invention. As a particularly preferable probeand primer in the detection of RNA in tissue, an RNA probe (riboprobe)can be exemplified.

The probe and the primer according to the present invention includethose comprising a nucleotide sequence consisting of at least 10,preferably at least 15 contiguous nucleotides of a nucleotide sequenceof a 187A5 gene, or a complementary sequence thereto. Also, the probeand the primer according to the present invention include thosecomprising a nucleotide sequence consisting of preferably 10 to 50 or 10to 30, more preferably 15 to 50 or 15 to 30, further preferably 20 to 50or 20 to 30, further more preferably 25 to 50 or 25 to 30, and mostpreferably 26 to 39 or 26 to 35 nucleotides.

The probe and the primer according to the present invention can be atleast 10 base length, preferably at least 15 base length, morepreferably at least 20 base length, and further preferably at least 25base length. The probe and the primer according to the present inventioncan also be preferably 10 to 50 base length or 10 to 30 base length,more preferably 15 to 50 base length or 15 to 30 base length, furtherpreferably 20 to 50 base length or 20 to 30 base length, further morepreferably 25 to 50 base length or 25 to 30 base length, and mostpreferably 26 to 39 base length or 26 to 35 base length.

According to preferable embodiments of the probe and the primeraccording to the present invention, there is provided a polynucleotidefor use in the detection or selection of a dopaminergic neuronprogenitor cell, and preferably a dopaminergic neuron proliferativeprogenitor cell in the mesencephalon, comprising a nucleotide sequenceconsisting of at least 10 (more preferably, at least 15) contiguousnucleotides of a nucleotide sequence of a 187A5 gene, or a complementarysequence thereto and having 15 to 50 base length or 15 to 30 baselength, more preferably 25 to 50 base length or 25 to 30 base length,and most preferably 26 to 39 base length or 26 to 35 base length, whichcan hybridize with a 187A5 gene.

According to preferable embodiments of the probe according to thepresent invention, there is also provided a polynucleotide that canhybridize to a high discrimination part in the nucleotide sequence ofthe 187A5 gene. By using such a polynucleotide, it becomes possible todetect the progenitor cells, and preferably the proliferative progenitorcells with higher accuracy. Such a polynucleotide includes apolynucleotide that hybridizes to a nucleotide sequence comprising apart or all of a nucleotide sequence of nucleotides 774 to 1221 or 2403to 2666 of SEQ ID NO: 1

According to preferable embodiments of the primer according to thepresent invention, there is also provided those that can amplify a highdiscrimination part in the nucleotide sequence of the 187A5 gene by anucleic acid amplification method, and a polynucleotide that canhybridize to the high discrimination part. By using such apolynucleotide, it becomes possible to detect the progenitor cells, andpreferably the proliferative progenitor cells with higher accuracy. Sucha polynucleotide includes a polynucleotide that can amplify, by anucleic acid amplification method, a nucleotide sequence comprising apart or all of a nucleotide sequence of nucleotides 774 to 1221 or 2403to 2666 of SEQ ID NO: 1.

The probe according to the present invention can be used as a probeaccording to the general methods in known methods for detecting a geneof interest, such as a northern blotting method, a southern blottingmethod or in situ hybridization method.

The probe according to the present invention can be chemicallysynthesized based on the nucleotide sequences disclosed in the presentspecification. The preparation of the probe is well-known and can beperformed, for example, according to “Molecular Cloning, A LaboratoryManual 2^(nd) ed.” (Cold Spring Harbor Press (1989)) or “CurrentProtocols in Molecular Biology” (John Wiley & Sons (1987-1997)).

The primer according to the present invention can also be used as aprimer set consisting of two or more primers according to the presentinvention.

The primer and the primer set according to the present invention can beused as a primer and a primer set according to the general methods inknown methods for detecting a gene of interest by using a nucleic acidamplification method such as a PCR method, a RT-PCR method, a real-timePCR method or in situ PCR.

The primer set according to the present invention can be selected sothat the nucleotide sequence of the 187A5 gene can be amplified by anucleic acid amplification method such as a PCR method. The nucleic acidamplification method is well-known, and selection of the primer set inthe nucleic acid amplification method is understood by those skilled inthe art. For example, in the PCR method, primers can be selected so thatone of two primers (primer pair) is paired with the plus strand of thedouble-strand DNA of the 187A5 gene while the other primer is pairedwith the minus strand of the double-strand DNA, and with a strandextended by one primer, the other primer can be paired. Moreover, in theLAMP method (WO 00/28082), with respect to the target gene, threeregions F3c, F2c and F1c and three regions B1, B2 and B3 are definedfrom the 3′ end side and from the 5′ end side, respectively, and byusing these six regions, four primers can be designed.

The primer according to the present invention can be chemicallysynthesized based on the nucleotide sequences disclosed in the presentspecification. The preparation of the primer is well-known and can beperformed, for example, according to “Molecular Cloning, A LaboratoryManual 2^(nd) ed.” (Cold Spring Harbor Press (1989)) or “CurrentProtocols in Molecular Biology” (John Wiley & Sons (1987-1997)).

[Antibody]

The antibody according to the present invention can specificallyrecognize a 187A5 protein. According to Example 5, it was confirmed thatthe 187A5 protein exists in dopaminergic neuron progenitor cells.Accordingly, the existence of the 187A5 protein is useful as an indexfor dopaminergic neuron progenitor cells including dopaminergic neuronproliferative progenitor cells. Therefore, the antibody according to thepresent invention can be used as a marker for detecting dopaminergicneuron progenitor cells, and preferably dopaminergic neuron progenitorcells.

The 187A5 protein is expressed on the cell surface in a directionwherein the N-terminal side thereof can be located in the extracellularspace (Example 4). Therefore, the antibody according to the presentinvention has the advantage that the dopaminergic neuron progenitorcells can be detected or selected as live cells (Example 6). Moreover,the antibody according to the present invention has the advantage thatES cell-derived cells can also be detected or selected (Example 7).

The 187A5 protein for obtaining the antibody according to the presentinvention may have antigenicity of 187A5 and includes theabove-described protein. Moreover, it includes a protein having an aminoacid sequence of the 187A5 protein in which one or more amino acidresidues are deleted, inserted, substituted or added. It is known thatin such a protein, the same biological activity as the original proteinis maintained (Mark et al. (1984) Proc. Natl. Acad. Sci. USA 81: 5662-6;Zoller and Smith (1982) Nucleic Acids Res. 10: 6487-500; Wang et al.(1984) Science 224: 1431-3; and Dalbadie-McFarland et al. (1982) Proc.Natl. Acad. Sci. USA 79: 6409-13). A method by which in a protein, oneor more amino acid residues are deleted, inserted, substituted or addedin the state of maintaining the antigenicity of the original protein isknown. For example, a polynucleotide encoding a mutant protein can beprepared by site-directed mutagenesis and can be appropriately expressedto obtain the protein (Molecular Cloning, A Laboratory Manual 2^(nd)ed., Cold Spring Harbor Press (1989); Current Protocols in MolecularBiology, John Wiley & Sons, (1987-1997), Section 8.1-8.5; Hashimoto-Gotoet al. (1995) Gene 152: 271-5; Kinkel (1985) Proc. Natl. Acad. Sci. USA82: 488-92; Kramer and Fritz (1987) Method. Enzymol 154: 350-67; andKunkel (1988) Method. Enzymol. 85: 2763-6).

The antibody according to the present invention also includes anantibody specific for a part of a 187A5 protein. Specifically, the 187A5protein for obtaining the antibody of the present invention includes apolypeptide having the full-length amino acid sequence of the 187A5protein as well as a polypeptide fragment having a sequence of at least6 amino acid residues or more (for example, 8, 10, 12 or 15 amino acidresidues or more) of the 187A5 protein. The polypeptide fragment of the187A5 protein in the present specification may be any fragment as longas having the 187A5 protein or antigenicity thereof.

Preferable fragments can include polypeptide fragments such as the aminoterminal of the 187A5 protein. The antigenic determinant site of thepolypeptide is estimated by a method of analyzinghydrophobicity/hydrophilicity of the amino acid sequence of the protein(Kyte-Doolittle (1982) J. Mol. Biol. 157: 105-22) or a method ofanalyzing the secondary structure (Chou-Fasman (1978) Ann. Rev. Biochem.47: 251-76), and furthermore, can be confirmed by a computer program(Anal. Biochem. 151: 540-6 (1985)) or a technique such as a PEPSCANmethod (Japanese Patent Laid-Open Publication No. 60-500684) ofsynthesizing a short peptide and confirming its antigenicity.

The antibody capable of binding to the 187A5 protein includes:

an antibody capable of binding to a protein consisting of the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 orSEQ ID NO: 10, or a part thereof;

an antibody capable of binding to a protein consisting of the amino acidsequence of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19or, or a part thereof;

an antibody capable of binding to a protein consisting of the amino acidsequence of SEQ ID NO: 22, or a part thereof;

an antibody capable of binding to a protein consisting of the amino acidsequence of SEQ ID NO: 24, or a part thereof;

an antibody capable of binding to a protein consisting of the amino acidsequence of SEQ ID NO: 26, or a part thereof; and

an antibody capable of binding to a protein consisting of the amino acidsequence of SEQ ID NO: 28, or a part thereof.

According to preferable embodiments of the antibody according to thepresent invention, there is provided an antibody that recognizes a highdiscrimination polypeptide region in the 187A5 protein. By using such anantibody, it becomes possible to detect the progenitor cells, andpreferably the proliferative progenitor cells with higher accuracy. Suchan antibody includes an antibody capable of binding to a proteincomprising a polypeptide consisting of at least 5 amino acid residues(preferably, at least 6 amino acid residues) or all of an amino acidsequence of amino acids 248 to 397 or 792 to 877 of SEQ ID NO: 2.

According to preferable embodiments of the antibody according to thepresent invention, there is also provided an antibody that recognizes apolypeptide region expressed in the extracellular space of the 187A5protein. By using such an antibody, it becomes possible to detect theprogenitor cells, and preferably the proliferative progenitor cells aslive cells. Such an antibody includes an antibody capable of binding tothe polypeptide region expressed in the extracellular space of the 187A5protein, for example, an antibody capable of binding to a proteincomprising a polypeptide consisting of at least 5 amino acid residues(preferably, at least 6 amino acid residues) or all of an amino acidsequence of amino acids 28 to 927 of SEQ ID NO: 2, amino acids 16 to1267 of SEQ ID NO: 4, amino acids 1 to 550 of SEQ ID NO: 6, amino acids1 to 542 of SEQ ID NO: 8, amino acids 1 to 418 of SEQ ID NO: 10, aminoacids 76 to 964 of SEQ ID NO: 12, amino acids 40 to 928 of SEQ ID NO:15, amino acids 1 to 540 of SEQ ID NO: 17, amino acids 40 to 1106 of SEQID NO: 19, amino acids 24 to 1524 of SEQ ID NO: 22, amino acids 43 to1018 of SEQ ID NO: 24, amino acids 43 to 908 of SEQ ID NO: 26 or aminoacids 1 to 866 of SEQ ID NO: 28.

The antibody according to the present invention can be obtained by usinga well-known method for those skilled in the art (for example, “CurrentProtocols in Molecular Biology” (John Wiley & Sons (1987)) andAntibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold SpringHarbor Laboratory (1988)).

The antibody according to the present invention includes a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a single-strandantibody (scFv), a humanized antibody, a polyspecific antibody andantibody fragments such as Fab, Fab′, F(ab′)₂, Fc and Fv.

In the case of the polyclonal antibody, the blood of a mammal in whichan antigen is sensitized is extracted, and serum can be segregated aspolyclonal antibody-containing serum from the blood by a known method.

According to need, fractions containing the polyclonal antibody can alsobe further isolated from this serum.

In the case of the monoclonal antibody, antibody-producing cellsobtained from the spleen or lymph node of the above-described mammal inwhich an antigen is sensitized are extracted and cell-fused with myelomacells or the like. The obtained hybridomas (fused cells) are cloned, andantibodies can be collected as the monoclonal antibody from the culturesthereof.

A fragment of the 187A5 protein can be used as the immunizing antigen.Alternatively, those synthesized based on the above-described amino acidsequence can be used. The antigen may be used as a complex with acarrier protein. For preparation of the complex of the antigen and thecarrier protein, various condensation agents such as glutaraldehyde,carbodiimide or maleimide-activated ester can be used. The carrierprotein may be one generally used such as bovine serum albumin,thyroglobulin or hemocyanin, and a method for coupling at a ratio of 1to 5 is generally used.

The animal to be immunized includes a mouse, a rat, a hamster, a guineapig, a rabbit, a cat, a dog, a pig, a goat, a horse and a bovine, and,preferably, includes a mouse, a rat, a rabbit, a guinea pig and ahamster. The injection method includes subcutaneous, muscular orintraperitoneal administration. In the administration, the antigen maybe mixed with complete Freund's adjuvant or incomplete Freund'sadjuvant. The administration is generally performed once per 2 to 5weeks.

The antibody-producing cells obtained from the spleen or lymph node ofthe immunized animal are cell-fused with myeloma cells and isolated ashybridomas. The myeloma cells to be used are derived from a mouse, arat, a human, or the like, and preferably, derived from the same speciesas the antibody-producing cells, but cells between different species areoccasionally possible.

Operation of the hybridomas (cell fusion) can be performed according toa previously known method, for example, the method disclosed in Nature,256, 495, 1975. Fusion accelerators include polyethylene glycol andSendai virus. In general, the cell fusion can be performed by reactionfor approximately 1 to 10 minutes so that the ratio between the numberof the antibody-producing cells and the number of the myeloma cells isgenerally approximately 1:1 to 10:1, under a temperature of 20 to 40°C., and preferably 30 to 37° C. by using polyethylene glycol (averagemolecular weight 1000 to 4000) having a concentration of approximately20 to 50%.

For screening of the antibody-producing hybridomas, variousimmunochemical methods can be used, which include an ELISA method byusing a microplate coated with the 187A5 protein, an EIA method by usinga microplate coated with an anti-immunoglobulin antibody, and animmunoblotting method by using a nitrocellulose transfer membrane afterelectrophoresing samples containing the 187A5 protein.

From such wells, cloning is further performed, for example, by alimiting dilution method, and thereby, clones can be obtained. Selectionand culture of the hybridomas are generally performed in a medium foranimal cells (for example, RPMI1640) containing 10 to 20% fetal bovineserum to which HAT (hypoxanthine, aminopterin and thymidine) is added.The clones obtained as described above are transplanted into theperitoneal cavity of an SCID mouse to which pristine is preliminarilyadministered, and ascitic fluid containing the monoclonal antibody at ahigh concentration is collected after 10 to 14 days, and can be used asa material for antibody purification. Also, the clones can be cultured,and the cultures thereof can also be used as a material for antibodypurification.

For the purification of the monoclonal antibody, a previously knownmethod as an immunoglobulin purification method may be used, and thepurification can be easily achieved, for example, by a means such as anammonium sulfate fraction method, a PEG fraction method, an ethanolfraction method, use of an anion exchanger, or affinity chromatographyusing the 187A5 protein.

The purification of the polyclonal antibody from the serum can besimilarly performed.

[Detection Method]

The expression of the 187A5 gene serves as an index for the existence ofdopaminergic neuron progenitor cells, and preferably dopaminergic neuronproliferative progenitor cells, as described above. Therefore, accordingto the present invention, the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected by detecting expression of a 187A5 gene.

The method for “detecting expression of a 187A5 gene” used herein is notparticularly limited as long as being capable of detecting theexpression of the 187A5 gene in cell samples to be tested, and can beperformed, for example, by the following steps of:

(a) contacting a cell sample to be tested, with the probe, the primer orthe primer set according to the present invention; and(b) detecting the presence or absence of reactivity.

The method for “detecting the presence or absence of reactivity” usedherein, for example, includes hybridization methods and nucleic acidamplification methods.

The “cell sample to be tested” used herein may be cell samples that arethought to contain the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells, and,preferably, cells in the mesencephalon ventral region can be used. Thecells in the mesencephalon ventral region can be obtained by a knownmethod (Studer, L., et al. Nature Neurosci (1998) 1: 290-295). Forexample, fetus's (preferably, human aborted fetus's) or patient's owncells of the mesencephalon ventral region can be used as the cell sampleto be tested. Moreover, culture cells containing dopaminergic neuronprogenitor cells, and preferably dopaminergic neuron proliferativeprogenitor cells induced to differentiate in vitro can be used. Theinduction to differentiate into the dopaminergic neuron progenitor cellsor the dopaminergic neuron proliferative progenitor cells in vitro canbe performed by differentiation treatment by a known method such as anSDIA method (Kawasaki et al. Neuron (2000) 28 (1): 31-40) or a 5-stagemethod (Lee, S H., et al. Nature Biotech (2000) 18: 675-579) using, as astarting material, cells such as known ES cells (Kawasaki et al. Neuron(2000) 28 (1): 31-40) and Lee, S H., et al. Nature Biotech (2000) 18:675-579), bone marrow stromal cells, nerve-derived immortalized celllines (Japanese Patent Laid-Open Publication No. 8-509215, No. 11-506930and No. 2002-522070) or neuron primordial cells (Japanese PatentLaid-Open Publication No. 11-509729). Preferably, ES cells subjected tothe differentiation treatment by the SDIA method can be used as the cellsample to be tested.

The “SDIA method” used herein can be performed by co-culturing ES cellsand the stromal cell line PA6 in a serum-free medium (Kawasaki et. al.Neuron. 2000 28 (1): 31-40). Moreover, the “5-stage method” can beperformed as follows. ES cells are cultured on a non-adherent cultureplate in the presence of serum, and thereby, an embryoid body (EB) isformed. Sequentially, the EB is attached onto an adherent culture plate,and thereby, neuron progenitor cells are selected. Finally, a growthfactor such as Shh, FGF2 or FGF8 is added thereto, and thereby,dopaminergic neuron progenitor cells are induced (Lee, S H., et al.Nature Biotech (2000) 18: 675-579).

According to the first embodiment of the detection method according tothe present invention, using the probe according to the presentinvention, the polynucleotide for detection hybridizes to a nucleic acidsample (mRNA or a transcript thereof), and the hybridization complex,namely, the nucleotide double strand, is detected. Thus, the expressionof the 187A5 gene can be detected in the cell sample.

For the detailed procedure of the hybridization method, there can bereferred to “Molecular Cloning, A Laboratory Manual 2^(nd) ed.” (ColdSpring Harbor Press (1989), particularly, Sections 9.47-9.58), “CurrentProtocols in Molecular Biology” (John Wiley & Sons (1987-1997),particularly, Sections 6.3-6.4), and “DNA Cloning 1: Core Techniques, APractical Approach 2^(nd) ed.” (Oxford University (1995), particularly,Section 2.10 for the conditions).

The detection of expression of a 187A5 gene by using the hybridizationmethod can be performed, for example, by the following steps of:

(a-1) contacting a polynucleotide derived from a cell sample to betested, with the probe according to the present invention; and(b-1) detecting a hybridization complex.

In step (a-1), mRNA prepared from the cell sample that is thought tocontain dopaminergic neuron progenitor cells, and preferablydopaminergic neuron proliferative progenitor cells, or a complementaryDNA (cDNA) transcribed from the mRNA can be contacted, as thepolynucleotide derived from the cell sample to be tested, with theprobe.

In the detection method by using the probe, the probe can be labeled.The label includes a label by using radioactivity (such as ³²P, ¹⁴C and³⁵S), fluorescence (such as FITC and europium), an enzyme (such asperoxidase or alkaline phosphatase) reaction such as chemical coloring,or the like.

The detection of the hybridization product can be performed by using awell-known method such as northern hybridization, southern hybridizationor colony hybridization.

The cells in which the hybridization complex is detected are thoseexpressing a 187A5 gene, and therefore, can be determined as thedopaminergic neuron progenitor cells, and preferably the dopaminergicneuron proliferative progenitor cells.

According to the second embodiment of the detection method according tothe present invention, using the primer or the primer set according tothe present invention, a nucleic acid sample (mRNA or a transcriptthereof) is amplified by a nucleic acid amplification method, and theamplification product is detected. Thus, the expression of the 187A5gene can be detected in the cell sample.

The detection of expression of a 187A5 gene by using the nucleic acidamplification method can be performed, for example, by the followingsteps of:

(a-2) performing a nucleic acid amplification method by using apolynucleotide derived from a cell sample to be tested as a template andthe primer or the primer set according to the present invention; and(b-2) detecting a formed amplification product.

In step (a-2), mRNA prepared from the sample that is thought to containdopaminergic neuron progenitor cells, and preferably dopaminergic neuronproliferative progenitor cells, or a complementary DNA (cDNA)transcribed from the mRNA can be used as the template.

The detection of the amplification product can be performed by using anucleic acid amplification method such as a PCR method, a RT-PCR method,a real-time PCR method or a LAMP method.

The cells in which the amplification product is detected are thoseexpressing a 187A5 gene, and therefore, can be determined as thedopaminergic neuron progenitor cells, and preferably the dopaminergicneuron proliferative progenitor cells.

The 187A5 protein serves as an index for the existence of dopaminergicneuron progenitor cells, and preferably dopaminergic neuronproliferative progenitor cells, as described above. Therefore, accordingto the present invention, the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected by detecting a 187A5 protein.

The method for “detecting a 187A5 protein” used herein is notparticularly limited as long as being capable of detecting the 187A5protein in cell samples to be tested, and, for example, includesantigen-antibody reaction methods.

According to the third embodiment of the detection method according tothe present invention, the antibody according to the present inventionand the cell sample are contacted, and the antigen-antibody reaction isdetected. Thus, the 187A5 protein can be detected in the cell sample.

The detection of a 187A5 protein by using the antigen-antibody reactioncan be performed, for example, by the following steps of:

(c) contacting a protein derived from a cell sample to be tested, withthe antibody according to the present invention; and(d) detecting the presence or absence of reactivity.

The method for “detecting the presence or absence of reactivity” usedherein, for example, includes antigen-antibody reaction methods.

The “cell sample to be tested” used herein can be cell samples to betested that are thought to contain the dopaminergic neuron progenitorcells, and preferably the dopaminergic neuron proliferative progenitorcells, and are preferably cells in the mesencephalon ventral region orculture cells containing dopaminergic neuron progenitor cells, andpreferably dopaminergic neuron proliferative progenitor cells induced todifferentiate in vitro. For example, those derived from an embryonicmesencephalon can be used as the cell sample to be tested. The methodfor obtaining the cell sample to be tested is as described above.

The detection of a 187A5 protein by using the antigen-antibody reactionmethod can be performed, for example, by the following steps of:

(c-1) contacting a protein derived from a cell sample to be tested, withthe antibody according to the present invention; and(d-1) detecting an antigen-antibody complex.

The method for detecting the antigen-antibody reaction is well-known forthose skilled in the art, and, for example, a 187A5 protein can bedetected in the cell sample to be tested that is thought to containdopaminergic neuron progenitor cells, and preferably dopaminergic neuronproliferative progenitor cells, by an immunological method. For theimmunological method, a previously known method such as animmunohistologic staining method, an enzyme-linked immunosorbent assay,a western blotting method, an agglutination method, a competition methodor a sandwich method, can be applied to the cell sample subjected toappropriate treatment according to need, such as cell separation orextraction operation. The immunohistologic staining method can beperformed by, for example, a direct method by using a labeled antibodyor an indirect method by using a labeled antibody capable of binding tothe antibody. For the labeling agent, a known labeling substance such asa fluorescent substance, a radioactive substance, an enzyme, a metal ora pigment can be used.

The protein derived from a cell sample to be tested is preferably apolypeptide comprising the extracellular region (namely, the N-terminalregion).

The cells in which the antigen-antibody complex is detected are thoseexpressing a 187A5 protein, and therefore, can be determined as thedopaminergic neuron progenitor cells, and preferably the dopaminergicneuron proliferative progenitor cells.

For use in the treatment of the Parkinson's disease, it is desirablethat the purity of the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells ishigh.

The accuracy of the detection or selection of the dopaminergic neuronprogenitor cells, and preferably the dopaminergic neuron proliferativeprogenitor cells can be enhanced by performing each of theabove-described detection steps not only once but repeatedly.

Therefore, according to the detection method according to the presentinvention, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with higher accuracy by performing the above-described steptwice or more.

Moreover, the accuracy of the detection or selection of the dopaminergicneuron progenitor cells, and preferably the dopaminergic neuronproliferative progenitor cells can be further enhanced by using togetherother marker genes, preferably a dopaminergic neuron proliferativeprogenitor cell marker gene other than the 187A5 gene.

Therefore, according to the detection method according to the presentinvention, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with higher accuracy by using together a dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene or aprotein thereof, a postmitotic dopaminergic neuron precursor cell markergene or a protein thereof, a dopaminergic neuron progenitor cell markergene other than the 187A5 gene or a protein thereof, or a maturedopaminergic neuron cell marker gene or a protein thereof, and detectingnot only expression of the 187A5 gene, or a protein thereof but alsoexpression of the above-described other marker genes, or the proteinsthereof.

Dopaminergic neuron-related marker genes selectively expressed in eachof differentiation stages are shown in FIG. 1.

In the detection method characterized in that the expression of the187A5 gene is detected, the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using together a dopaminergicneuron proliferative progenitor cell marker gene other than the 187A5gene or a protein thereof, and detecting not only the 187A5 gene butalso expression of the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene, or the protein thereof.

Specifically, in step (a), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the expression of the dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, orthe protein thereof is detected. In this case, the cells in whichreactivity is detected (for example, the cells in which thehybridization complex or the amplification product is detected) in step(b) are those which express the 187A5 gene, and which express thedopaminergic neuron proliferative progenitor cell marker gene other thanthe 187A5 gene or which have the existence of the protein thereof. Thus,the cells can be determined as the detected or selected dopaminergicneuron progenitor cells, and preferably the detected or selecteddopaminergic neuron proliferative progenitor cells with high accuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-1) detecting expression of a dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, ora protein thereof, with respect to the cells in which reactivity isdetected (for example, the cells in which the hybridization complex orthe amplification product is detected) in step (b). In this case, instep (e-1), the cells in which the expression of the dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, orthe protein thereof is detected are those which express the 187A5 gene,and which express the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene or which have the existence of theprotein thereof. Thus, the cells can be determined as the detected orselected dopaminergic neuron progenitor cells, and preferably thedetected or selected dopaminergic neuron proliferative progenitor cellswith high accuracy.

In the detection method characterized in that the expression of the187A5 gene is detected, by using together a postmitotic dopaminergicneuron precursor cell marker gene or a protein thereof, it can beconfirmed that the 187A5 gene is expressed but the expression of thepostmitotic dopaminergic neuron precursor cell marker gene, or theprotein thereof is not detected. Thus, the dopaminergic neuronprogenitor cells, and preferably the dopaminergic neuron proliferativeprogenitor cells can be detected or selected with high accuracy.

Specifically, in step (a), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the expression of the postmitoticdopaminergic neuron precursor cell marker gene, or the protein thereofis not detected. In this case, the cells in which reactivity is detected(for example, the cells in which the hybridization complex or theamplification product is detected) in step (b) are those which expressthe 187A5 gene, which do not express the postmitotic dopaminergic neuronprecursor cell marker gene, and which do not have the existence of theprotein thereof. Thus, the cells can be determined as the detected orselected dopaminergic neuron progenitor cells, and preferably thedetected or selected dopaminergic neuron proliferative progenitor cellswith high accuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-2) detecting expression of a postmitotic dopaminergicneuron precursor cell marker gene, or a protein thereof, with respect tothe cells in which reactivity is detected (for example, the cells inwhich the hybridization complex or the amplification product isdetected) in step (b). In this case, in step (e-2), the cells in whichthe expression of the postmitotic dopaminergic neuron precursor cellmarker gene, or the protein thereof is not detected are those whichexpress the 187A5 gene, which do not express the postmitoticdopaminergic neuron precursor cell marker gene, and which do not havethe existence of the protein thereof. Thus, the cells can be determinedas the detected or selected dopaminergic neuron progenitor cells, andpreferably the detected or selected dopaminergic neuron proliferativeprogenitor cells with high accuracy.

In the detection method characterized in that the expression of the187A5 gene is detected, the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using together a dopaminergicneuron progenitor cell marker gene other than the 187A5 gene or aprotein thereof, and detecting not only the 187A5 gene but alsoexpression of the dopaminergic neuron progenitor cell marker gene otherthan the 187A5 gene, or the protein thereof.

Specifically, in step (a), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the expression of the dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof is detected. In this case, the cells in which reactivity isdetected (for example, the cells in which the hybridization complex orthe amplification product is detected) in step (b) are those whichexpress the 187A5 gene, and which express the dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene or which have theexistence of the protein thereof. Thus, the cells can be determined asthe detected or selected dopaminergic neuron progenitor cells, andpreferably the detected or selected dopaminergic neuron proliferativeprogenitor cells with high accuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-3) detecting expression of a dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene, or a proteinthereof, with respect to the cells in which reactivity is detected (forexample, the cells in which the hybridization complex or theamplification product is detected) in step (b). In this case, in step(e-3), the cells in which the expression of the dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof is detected are those which express the 187A5 gene, and whichexpress the dopaminergic neuron progenitor cell marker gene other thanthe 187A5 gene or which have the existence of the protein thereof. Thus,the cells can be determined as the detected or selected dopaminergicneuron progenitor cells, and preferably the detected or selecteddopaminergic neuron proliferative progenitor cells with high accuracy.

In the detection method characterized in that the expression of the187A5 gene is detected, by using together a mature dopaminergic neuroncell marker gene or a protein thereof, it can be confirmed that the187A5 gene is expressed but the expression of the mature dopaminergicneuron cell marker gene, or the protein thereof is not detected. Thus,the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy.

Specifically, in step (a), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the expression of the mature dopaminergicneuron cell marker gene, or the protein thereof is not detected. In thiscase, the cells in which reactivity is detected (for example, the cellsin which the hybridization complex or the amplification product isdetected) in step (b) are those which express the 187A5 gene, which donot express the mature dopaminergic neuron cell marker gene, and whichdo not have the existence of the protein thereof. Thus, the cells can bedetermined as the detected or selected dopaminergic neuron progenitorcells, and preferably the detected or selected dopaminergic neuronproliferative progenitor cells with high accuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-4) detecting expression of a mature dopaminergic neuroncell marker gene, or a protein thereof, with respect to the cells inwhich reactivity is detected (for example, the cells in which thehybridization complex or the amplification product is detected) in step(b). In this case, in step (e-4), the cells in which the expression ofthe mature dopaminergic neuron cell marker gene, or the protein thereofis not detected are those which express the 187A5 gene, which do notexpress the mature dopaminergic neuron cell marker gene, and which donot have the existence of the protein thereof. Thus, the cells can bedetermined as the detected or selected dopaminergic neuron progenitorcells, and preferably the detected or selected dopaminergic neuronproliferative progenitor cells with high accuracy.

In the detection method characterized in that the 187A5 protein isdetected, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by using together a dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene or aprotein thereof, and detecting not only the 187A5 protein but alsoexpression of the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene, or the protein thereof.

Specifically, in step (c), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the expression of the dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, orthe protein thereof is detected. In this case, the cells in whichreactivity is detected (for example, the cells in which theantigen-antibody complex is detected) in step (d) are those which havethe existence of the 187A5 protein, and which express the dopaminergicneuron proliferative progenitor cell marker gene other than the 187A5gene or which have the existence of the protein thereof. Thus, the cellscan be determined as the detected or selected dopaminergic neuronprogenitor cells, and preferably the detected or selected dopaminergicneuron proliferative progenitor cells with high accuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-1) detecting expression of a dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, ora protein thereof, with respect to the cells in which reactivity isdetected (for example, the cells in which the antigen-antibody complexis detected) in step (d). In this case, in step (e-1), the cells inwhich the expression of the dopaminergic neuron proliferative progenitorcell marker gene other than the 187A5 gene, or the protein thereof isdetected are those which have the existence of the 187A5 protein, andwhich express the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene or which have the existence of theprotein thereof. Thus, the cells can be determined as the detected orselected dopaminergic neuron progenitor cells, and preferably thedetected or selected dopaminergic neuron proliferative progenitor cellswith high accuracy.

In the detection method characterized in that the 187A5 protein isdetected, by using together a postmitotic dopaminergic neuron precursorcell marker gene or a protein thereof, it can be confirmed that the187A5 protein is expressed but the expression of the postmitoticdopaminergic neuron precursor cell marker gene, or the protein thereofis not detected. Thus, the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy.

Specifically, in step (c), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the expression of the postmitoticdopaminergic neuron precursor cell marker gene, or the protein thereofis not detected. In this case, the cells in which reactivity is detected(for example, the cells in which the antigen-antibody complex isdetected) in step (d) are those which have the existence of the 187A5protein, which do not express the postmitotic dopaminergic neuronprecursor cell marker gene, and which do not have the existence of theprotein thereof. Thus, the cells can be determined as the detected orselected dopaminergic neuron progenitor cells, and preferably thedetected or selected dopaminergic neuron proliferative progenitor cellswith high accuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-2) detecting expression of a postmitotic dopaminergicneuron precursor cell marker gene, or a protein thereof, with respect tothe cells in which reactivity is detected (for example, the cells inwhich the antigen-antibody complex is detected) in step (d). In thiscase, in step (e-2), the cells in which the expression of thepostmitotic dopaminergic neuron precursor cell marker gene, or theprotein thereof is not detected are those which have the existence ofthe 187A5 protein, which do not express the postmitotic dopaminergicneuron precursor cell marker gene, and which do not have the existenceof the protein thereof. Thus, the cells can be determined as thedetected or selected dopaminergic neuron progenitor cells, andpreferably the detected or selected dopaminergic neuron proliferativeprogenitor cells with high accuracy.

In the detection method characterized in that the 187A5 protein isdetected, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by using together a dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene or a proteinthereof, and detecting not only the 187A5 protein but also expression ofthe dopaminergic neuron progenitor cell marker gene other than the 187A5gene, or the protein thereof.

Specifically, in step (c), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the dopaminergic neuron progenitor cellmarker gene other than the 187A5 gene, or the protein thereof isdetected. In this case, the cells in which reactivity is detected (forexample, the cells in which the antigen-antibody complex is detected) instep (d) are those which have the existence of the 187A5 protein, andwhich express the dopaminergic neuron progenitor cell marker gene otherthan the 187A5 gene or which have the existence of the protein thereof.Thus, the cells can be determined as the detected or selecteddopaminergic neuron progenitor cells, and preferably the detected orselected dopaminergic neuron proliferative progenitor cells with highaccuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-3) detecting expression of a dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene, or a proteinthereof, with respect to the cells in which reactivity is detected (forexample, the cells in which the antigen-antibody complex is detected) instep (d). In this case, in step (e-3), the cells in which the expressionof the dopaminergic neuron progenitor cell marker gene other than the187A5 gene, or the protein thereof is detected are those which have theexistence of the 187A5 protein, and which express the dopaminergicneuron progenitor cell marker gene other than the 187A5 gene or whichhave the existence of the protein thereof. Thus, the cells can bedetermined as the detected or selected dopaminergic neuron progenitorcells, and preferably the detected or selected dopaminergic neuronproliferative progenitor cells with high accuracy.

In the detection method characterized in that the 187A5 protein isdetected, by using together a mature dopaminergic neuron cell markergene or a protein thereof, it can be confirmed that the 187A5 protein isexpressed but the expression of the mature dopaminergic neuron cellmarker gene, or the protein thereof is not detected. Thus, thedopaminergic neuron progenitor cells, and preferably the dopaminergicneuron proliferative progenitor cells can be detected or selected withhigh accuracy.

Specifically, in step (c), the dopaminergic neuron progenitor cells, andpreferably the dopaminergic neuron proliferative progenitor cells can bedetected or selected with high accuracy by using, as the cell sample tobe tested, the cells in which the expression of the mature dopaminergicneuron cell marker gene, or the protein thereof is not detected. In thiscase, the cells in which reactivity is detected (for example, the cellsin which the antigen-antibody complex is detected) in step (d) are thosewhich have the existence of the 187A5 protein, which do not express themature dopaminergic neuron cell marker gene, and which do not have theexistence of the protein thereof. Thus, the cells can be determined asthe detected or selected dopaminergic neuron progenitor cells, andpreferably the detected or selected dopaminergic neuron proliferativeprogenitor cells with high accuracy.

Moreover, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with high accuracy by performing the method further comprisingthe step of (e-4) detecting expression of a mature dopaminergic neuroncell marker gene, or a protein thereof, with respect to the cells inwhich reactivity is detected (for example, the cells in which theantigen-antibody complex is detected) in step (d). In this case, in step(e-4), the cells in which the expression of the mature dopaminergicneuron cell marker gene, or the protein thereof is not detected arethose which have the existence of the 187A5 protein, which do notexpress the mature dopaminergic neuron cell marker gene, and which donot have the existence of the protein thereof. Thus, the cells can bedetermined as the detected or selected dopaminergic neuron progenitorcells, and preferably the detected or selected dopaminergic neuronproliferative progenitor cells with high accuracy.

“The dopaminergic neuron proliferative progenitor cell marker gene otherthan the 187A5 gene or the protein thereof” is a “dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene” ora “dopaminergic neuron proliferative progenitor cell marker proteinother than the 187A5 protein”.

“The dopaminergic neuron proliferative progenitor cell marker gene otherthan the 187A5 gene” includes a dopaminergic neuron proliferativeprogenitor cell marker gene other than the 187A5 gene which is expressedin the mesencephalon most ventral ventricular zone (VZ region), andincludes an Lrp4 gene, a Nato3 gene, an Msx1 gene, an Msx2 gene and aMash1 gene.

The Lrp4 gene is described in WO 2004/065599. The Nato3 gene isdescribed in WO 2007/021003. The Msx1 gene and the Msx2 gene aredescribed in WO 2007/021004. The Mash1 gene is described in Kele J,Simplicio N, Ferri A L, Mira H, Guillemot F, Arenas E, Ang S L.Neurogenin 2 is required for the development of ventral mesencephalondopaminergic neurons. Development. 2006 February; 133 (3): 495-505.

The detection of the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene is not particularly limited aslong as using a method by which expression of the known gene can bedetected, and, for example, includes the hybridization method and thenucleic acid amplification method, as described above.

“The dopaminergic neuron proliferative progenitor cell marker proteinother than the 187A5 protein” includes a dopaminergic neuronproliferative progenitor cell marker protein other than the 187A5protein which is expressed in the mesencephalon most ventral ventricularzone (VZ region), and, preferably, includes a protein detected only indopaminergic neuron proliferative progenitor cells.

Such a protein includes proteins of an Lrp4 gene, a Nato3 gene, an Msx1gene, an Msx2 gene and a Mash1 gene.

The detection of the dopaminergic neuron proliferative progenitor cellmarker protein other than the 187A5 protein is not particularly limitedas long as using a method by which expression of the known protein canbe detected, and, for example, includes the antigen-antibody reactionmethod, as described above.

“The postmitotic dopaminergic neuron precursor cell marker gene or theprotein thereof” includes a gene expressed in the mesencephalon mostventral mantle layer (ML region) or a protein thereof, and includes aNurr1 gene, an En1 gene, an En2 gene, a Ptx3 gene and a TH gene.Moreover, the marker gene includes a gene expressed in the mesencephalonmost ventral ventricular zone (VZ region) or a protein thereof, andincludes a 65B13 gene.

The Nurr1 gene is described in Science. 1997 11; 276 (5310): 248-50. TheEn1 gene is described in J. Neurosci. 2001 21 (9): 3126-34. The En2 geneis described in J. Neurosci. 2001 21 (9): 3126-34. The Ptx3 gene isdescribed in Proc. Natl. Acad. Sci. 1997 94: 13305-10. The TH gene isdescribed in Science. 1997 11; 276 (5310): 248-50. The 65B13 gene isdescribed in WO 2004/038018.

The detection of the postmitotic dopaminergic neuron precursor cellmarker gene or the protein thereof is not particularly limited as longas using a method by which expression of the known gene or the proteinthereof can be detected, and, for example, includes the hybridizationmethod, the nucleic acid amplification method and the antigen-antibodyreaction method, as described above.

“The dopaminergic neuron progenitor cell marker gene other than the187A5 gene or the protein thereof” is a “dopaminergic neuron progenitorcell marker gene other than the 187A5 gene” or a “dopaminergic neuronprogenitor cell marker protein other than the 187A5 protein”.

“The dopaminergic neuron progenitor cell marker gene other than the187A5 gene” includes a dopaminergic neuron progenitor cell marker geneother than the 187A5 gene which is expressed in the mesencephalon mostventral region, and includes an Lmx1a gene.

The Lmx1a gene is described in WO 2005/052190.

The detection of the dopaminergic neuron progenitor cell marker geneother than the 187A5 gene is not particularly limited as using a methodby which expression of the known gene can be detected, and, for example,includes the hybridization method and the nucleic acid amplificationmethod, as described above.

“The dopaminergic neuron progenitor cell marker protein other than the187A5 protein” includes a dopaminergic neuron progenitor cell markerprotein other than the 187A5 protein which is expressed in themesencephalon most ventral region. Such a protein includes a protein ofan Lmx1a gene.

The detection of the dopaminergic neuron progenitor cell marker proteinother than the 187A5 protein is not particularly limited as long asusing a method by which expression of the known protein can be detected,and, for example, includes the antigen-antibody reaction method, asdescribed above.

“The mature dopaminergic neuron cell marker gene” includes a DAT gene.

The DAT gene is described in Development 2003 131: 1145-55.

The detection of the mature dopaminergic neuron cell marker gene or theprotein thereof is not particularly limited as long as using a method bywhich expression of the known gene or the protein thereof can bedetected, and, for example, includes the hybridization method, thenucleic acid amplification method and the antigen-antibody reactionmethod, as described above.

Moreover, the accuracy of the detection or selection of the dopaminergicneuron progenitor cells, and preferably the dopaminergic neuronproliferative progenitor cells can be further enhanced by using togethera vector comprising a gene construct in which a promoter of the 187A5gene is operably linked to a marker gene.

Therefore, according to the detection method according to the presentinvention, the dopaminergic neuron progenitor cells, and preferably thedopaminergic neuron proliferative progenitor cells can be detected orselected with higher accuracy by using together a gene construct inwhich a promoter of the 187A5 gene is operably linked to a marker gene,and detecting not only expression of the 187A5 gene, or the proteinthereof but also expression of the marker gene.

The detection of the dopaminergic neuron progenitor cells by using thevector comprising a gene construct in which a promoter of the 187A5 geneis operably linked to a marker gene can be performed, for example,according to Japanese Patent Laid-Open Publication No. 2002-51775.

A marker gene that can be detected under the control of apromoter/enhancer of the 187A5 gene expressed in dopaminergic neuronprogenitor cells is introduced into each cell in a cell population, andthe expression of the marker gene is detected. Thus, the dopaminergicneuron progenitor cells can be detected.

Specifically, the dopaminergic neuron progenitor cells can be detectedor selected by performing the steps of transforming the cell sample tobe tested, with a vector comprising a gene construct in which a promoterof the gene according to the present invention is operably linked to amarker gene, and detecting expression of the marker gene in the cellsample to be tested. In this case, in the step, the cells in which theexpression of the marker gene is detected can be determined as thedetected or selected dopaminergic neuron progenitor cells, andpreferably the detected or selected dopaminergic neuron proliferativeprogenitor cell with high accuracy.

The nucleotide sequence of the “promoter of the gene according to thepresent invention” used herein includes a nucleotide sequence of apromoter region obtained by expression region analysis of the 187A5 geneto be described later, and also includes a modified sequence thereofhaving approximately equivalent promoter activity.

The “marker gene” used herein may be a marker gene that can be detectedunder the control of a promoter/enhancer of the 187A5 gene, and includesGFP.

The “gene construct” used herein may have a structure in which the 187A5gene is linked upstream or downstream of the marker gene under thecontrol of an expression control sequence (including a promoter, anenhancer, or the like) of the 187A5 gene. In addition, a gene encodingthe maker can be knocked in to the 187A5 gene locus. As preferableembodiments of the gene construct, constructs having structuresschematically described in 2 to 4 in FIG. 15 can be exemplified.

[Detection Kit]

The present invention provides a detection kit for performing thedetection method according to the present invention.

The first embodiment of the detection kit according to the presentinvention includes a detection kit for performing the first embodimentof the detection method according to the present invention, andspecifically, includes a kit for detecting expression of a 187A5 gene,comprising at least the probe according to the present invention. Thisprobe may be labeled. The detection kit detects the expression of the187A5 gene by a hybrid formation method. Therefore, the detection kit ofthe first embodiment can optionally further include various reagents forperforming the hybrid formation method, for example, a substratecompound for use in the detection of a label, a hybridization buffer,instructions, equipment, and/or so forth.

Moreover, a detection kit for performing the detection with highaccuracy includes the kit further comprising a probe, a primer, a primerset or an antibody which can detect expression of a dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, ora protein thereof, expression of a postmitotic dopaminergic neuronprecursor cell marker gene, or a protein thereof, expression of adopaminergic neuron progenitor cell marker gene other than the 187A5gene, or a protein thereof, or expression of a mature dopaminergicneuron cell marker gene, or a protein thereof. The probe, the primer,the primer set or the antibody may be labeled. By any of the hybridformation method, the nucleic acid amplification method and theantigen-antibody reaction method, the detection kit further detects theexpression of the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene, or the protein thereof, theexpression of the postmitotic dopaminergic neuron precursor cell markergene, or the protein thereof, the expression of the dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof, or the expression of the mature dopaminergic neuron cell markergene, or the protein thereof.

The second embodiment of the detection kit according to the presentinvention includes a detection kit for performing the second embodimentof the detection method according to the present invention, andspecifically, includes a kit for detecting expression of a 187A5 gene,comprising at least the primer according to the present invention or theprimer set according to the present invention. The detection kit detectsthe expression of the 187A5 gene by the nucleic acid amplificationmethod. Therefore, the detection kit of the second embodiment canoptionally further include various reagents for performing the nucleicacid amplification method, for example, a buffer, an internal standardindicating that the amplification reaction can normally progress,instructions, equipment, and/or so forth.

Moreover, a detection kit for performing the detection with highaccuracy includes the kit further comprising a probe, a primer, a primerset or an antibody which can detect expression of a dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, ora protein thereof, expression of a postmitotic dopaminergic neuronprecursor cell marker gene, or a protein thereof, expression of adopaminergic neuron progenitor cell marker gene other than the 187A5gene, or a protein thereof, or expression of a mature dopaminergicneuron cell marker gene, or a protein thereof. The probe, the primer,the primer set or the antibody may be labeled. By any of the hybridformation method, the nucleic acid amplification method and theantigen-antibody reaction method, the detection kit further detects theexpression of the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene, or the protein thereof, theexpression of the postmitotic dopaminergic neuron precursor cell markergene, or the protein thereof, the expression of the dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof, or the expression of the mature dopaminergic neuron cell markergene, or the protein thereof.

The third embodiment of the detection kit according to the presentinvention includes a detection kit for performing the third embodimentof the detection method according to the present invention, andspecifically, includes a kit for detecting a 187A5 protein, comprisingat least the antibody according to the present invention. This antibodymay be labeled. The detection kit detects the expression of the 187A5protein by detecting the antigen-antibody reaction. Therefore, thedetection kit of the third embodiment can optionally further includevarious reagents for performing the antigen-antibody reaction, forexample, a secondary antibody for use in an ELISA method or the like, acoloring reagent, a buffer, instructions, equipment, and/or so forth.

Moreover, a detection kit for performing the detection with highaccuracy includes the kit further comprising a probe, a primer, a primerset or an antibody which can detect expression of a dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, ora protein thereof, expression of a postmitotic dopaminergic neuronprecursor cell marker gene, or a protein thereof, expression of adopaminergic neuron progenitor cell marker gene other than the 187A5gene, or a protein thereof, or expression of a mature dopaminergicneuron cell marker gene, or a protein thereof. The probe, the primer,the primer set or the antibody may be labeled. By any of the hybridformation method, the nucleic acid amplification method and theantigen-antibody reaction method, the detection kit further detects theexpression of the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene, or the protein thereof, theexpression of the postmitotic dopaminergic neuron precursor cell markergene, or the protein thereof, the expression of the dopaminergic neuronprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof, or the expression of the mature dopaminergic neuron cell markergene, or the protein thereof.

Furthermore, a detection kit for performing the detection with highaccuracy includes the detection kits of the first to third embodimentaccording to the present invention, further comprising a vectorcomprising a gene construct in which a promoter of the 187A5 gene isoperably linked to a marker gene.

[Agent for Detection]

The present invention provides an agent for detection for performing thedetection method according to the present invention.

The first embodiment of the agent for detection according to the presentinvention includes an agent for detection for performing the firstembodiment of the detection method according to the present invention,and specifically, includes an agent for detecting expression of a 187A5gene, comprising at least the probe according to the present invention.This probe may be labeled. The agent for detection detects theexpression of the 187A5 gene by a hybrid formation method. Therefore,the agent for detection of the first embodiment can optionally furtherincludes various reagents for performing the hybrid formation method,for example, a substrate compound for use in the detection of a label, ahybridization buffer, instructions, equipment, and/or so forth.

Moreover, an agent for detection for performing the detection with highaccuracy includes the agent for detection further comprising a probe, aprimer, a primer set or an antibody which can detect expression of adopaminergic neuron proliferative progenitor cell marker gene other thanthe 187A5 gene, or a protein thereof, expression of a postmitoticdopaminergic neuron precursor cell marker gene, or a protein thereof,expression of a dopaminergic neuron progenitor cell marker gene otherthan the 187A5 gene, or a protein thereof, or expression of a maturedopaminergic neuron cell marker gene, or a protein thereof. The probe,the primer, the primer set or the antibody may be labeled. By any of thehybrid formation method, the nucleic acid amplification method and theantigen-antibody reaction method, the agent for detection furtherdetects the expression of the dopaminergic neuron proliferativeprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof, the expression of the postmitotic dopaminergic neuron precursorcell marker gene, or the protein thereof, the expression of thedopaminergic neuron progenitor cell marker gene other than the 187A5gene, or the protein thereof, or the expression of the maturedopaminergic neuron cell marker gene, or the protein thereof.

The second embodiment of the agent for detection according to thepresent invention includes an agent for detection for performing thesecond embodiment of the detection method according to the presentinvention, and specifically, includes an agent for detecting expressionof a 187A5 gene, comprising at least the primer according to the presentinvention or the primer set according to the present invention. Theagent for detection detects the expression of the 187A5 gene by thenucleic acid amplification method. Therefore, the agent for detection ofthe second embodiment can optionally further include various reagentsfor performing the nucleic acid amplification method, for example, abuffer, an internal standard indicating that the amplification reactioncan normally progress, instructions, equipment, and/or so forth

Moreover, an agent for detection for performing the detection with highaccuracy includes the agent for detection further comprising a probe, aprimer, a primer set or an antibody which can detect expression of adopaminergic neuron proliferative progenitor cell marker gene other thanthe 187A5 gene, or a protein thereof, expression of a postmitoticdopaminergic neuron precursor cell marker gene, or a protein thereof,expression of a dopaminergic neuron progenitor cell marker gene otherthan the 187A5 gene, or a protein thereof, or expression of a maturedopaminergic neuron cell marker gene, or a protein thereof. The probe,the primer, the primer set or the antibody may be labeled. By any of thehybrid formation method, the nucleic acid amplification method and theantigen-antibody reaction method, the agent for detection furtherdetects the expression of the dopaminergic neuron proliferativeprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof, the expression of the postmitotic dopaminergic neuron precursorcell marker gene, or the protein thereof, the expression of thedopaminergic neuron progenitor cell marker gene other than the 187A5gene, or the protein thereof, or the expression of the maturedopaminergic neuron cell marker gene, or the protein thereof.

The third embodiment of the agent for detection according to the presentinvention includes an agent for detection for performing the thirdembodiment of the detection method according to the present invention,and specifically, includes a agent for detecting a 187A5 protein,comprising at least the antibody according to the present invention.This antibody may be labeled. The agent for detection detects theexpression of the 187A5 protein by detecting the antigen-antibodyreaction. Therefore, the agent for detection of the third embodiment canoptionally further include various reagents for performing theantigen-antibody reaction, for example, a secondary antibody for use inan ELISA method or the like, a coloring reagent, a buffer, instructions,equipment, and/or so forth.

Moreover, an agent for detection for performing the detection with highaccuracy includes the agent for detection further comprising a probe, aprimer, a primer set or an antibody which can detect expression of adopaminergic neuron proliferative progenitor cell marker gene other thanthe 187A5 gene, or a protein thereof, expression of a postmitoticdopaminergic neuron precursor cell marker gene, or a protein thereof,expression of a dopaminergic neuron progenitor cell marker gene otherthan the 187A5 gene, or a protein thereof, or expression of a maturedopaminergic neuron cell marker gene, or a protein thereof. The probe,the primer, the primer set or the antibody may be labeled. By any of thehybrid formation method, the nucleic acid amplification method and theantigen-antibody reaction method, the agent for detection furtherdetects the expression of the dopaminergic neuron proliferativeprogenitor cell marker gene other than the 187A5 gene, or the proteinthereof, the expression of the postmitotic dopaminergic neuron precursorcell marker gene, or the protein thereof, the expression of thedopaminergic neuron progenitor cell marker gene other than the 187A5gene, or the protein thereof, or the expression of the maturedopaminergic neuron cell marker gene, or the protein thereof.

Furthermore, an agent for detection for performing the detection withhigh accuracy includes the agents for detection of the first to thirdembodiment according to the present invention, further comprising avector comprising a gene construct in which a promoter of the 187A5 geneis operably linked to a marker gene.

[Screening Method]

The detection method according to the present invention can be appliedto screening for an effective substance for inducing differentiationinto a dopaminergic neuron progenitor cell. Specifically, whether or notthe addition of a candidate substance has induced the differentiationinto a dopaminergic neuron progenitor cell, and preferably adopaminergic neuron proliferative progenitor cell is determined by usingexpression of a 187A5 gene, or a protein thereof as an index, andthereby, the effective substance for inducing differentiation into adopaminergic neuron progenitor cell can be screened for.

Therefore, the present invention provides a method for screening for aneffective substance for inducing differentiation into a dopaminergicneuron progenitor cell, comprising the following steps of:

(i) contacting a cell that can differentiate into a dopaminergic neuronprogenitor cell, with a substance to be tested; and(ii) detecting expression of a 187A5 gene, or a protein thereof in thecell that has been contacted with the substance to be tested.

The cell that can differentiate into a dopaminergic neuron progenitorcell in step (i) is preferably a cell that can differentiate into adopaminergic neuron proliferative progenitor cell, and can be preferablycollected from an embryonic mesencephalon or from culture cellscontaining neuron progenitor cells induced to differentiate from EScells.

“Contacting with a substance to be tested” in step (i) can be performed,for example, by adding the substance to be tested to culture cellscontaining the cell that can differentiate into a dopaminergic neuronprogenitor cell, and preferably a dopaminergic neuron proliferativeprogenitor cell.

The “substance to be tested” includes a synthesized low-molecularcompound, a protein, a synthesized peptide, a purified or partiallypurified polypeptide, an antibody, a bacterium-releasing material(including bacterial metabolites) and a nucleic acid (such as antisense,ribozyme and RNAi), and is preferably a compound or a salt thereof, or asolvate thereof (for example, a hydrate), but is not limited thereto.The “substance to be tested” may be a novel substance or a knownsubstance.

In step (ii), according to the detection method according to the presentinvention, the expression of the 187A5 gene, or the protein thereof canbe detected.

Specifically, steps (a-1) and (b-1) are performed for the detection byusing the hybridization method. Steps (a-2) and (b-2) are performed forthe detection by using the nucleic acid amplification method. Steps(c-1) and (d-1) are performed for the detection by using theantigen-antibody reaction method. Thus, the expression of the 187A5gene, or the protein thereof can be detected.

In step (ii), when the expression of the 187A5 gene, or the proteinthereof is detected in the cell sample to be tested by contacting thesubstance to be tested, the substance can be determined as the effectivesubstance for inducing differentiation into a dopaminergic neuronprogenitor cell, and preferably a dopaminergic neuron proliferativeprogenitor cell.

The substance specified by the screening method according to the presentinvention can be used as the effective substance for inducingdifferentiation into a dopaminergic neuron progenitor cell, andpreferably a dopaminergic neuron proliferative progenitor cell.

The present invention provides the method for screening for an effectivesubstance for inducing differentiation into a dopaminergic neuronprogenitor cell, further comprising the following step of:

(iii-1) detecting expression of a dopaminergic neuron proliferativeprogenitor cell marker gene other than the 187A5 gene, or a proteinthereof in the cell that has been contacted with the substance to betested.

When the expression of the 187A5 gene, or the protein thereof isdetected in step (ii), and the expression of the dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene, orthe protein thereof is detected in step (iii-1), the substance can bedetermined as the effective substance for inducing differentiation intoa dopaminergic neuron progenitor cell, and preferably a dopaminergicneuron proliferative progenitor cell, with high accuracy.

Step (iii-1) may be performed after step (i) and may be performed beforeor after step (ii).

The present invention provides the method for screening for an effectivesubstance for inducing differentiation into a dopaminergic neuronprogenitor cell, further comprising the following step of:

(iii-2) detecting expression of a postmitotic dopaminergic neuronprecursor cell marker gene, or a protein thereof in the cell that hasbeen contacted with the substance to be tested.

When the expression of the 187A5 gene, or the protein thereof isdetected in step (ii), but the postmitotic dopaminergic neuron precursorcell marker gene or the protein thereof is not detected in step (iii-2),the substance can be determined as the effective substance for inducingdifferentiation into a dopaminergic neuron progenitor cell, andpreferably a dopaminergic neuron proliferative progenitor cell, withhigh accuracy.

Step (iii-2) may be performed after step (i) and may be performed beforeor after step (ii).

“The dopaminergic neuron proliferative progenitor cell marker gene otherthan the 187A5 gene or the protein thereof” is a “dopaminergic neuronproliferative progenitor cell marker gene other than the 187A5 gene” ora “dopaminergic neuron proliferative progenitor cell marker proteinother than the 187A5 protein”.

“The dopaminergic neuron proliferative progenitor cell marker gene otherthan the 187A5 gene” includes a dopaminergic neuron proliferativeprogenitor cell marker gene other than the 187A5 gene which is expressedin the mesencephalon most ventral ventricular zone (VZ region), andincludes an Lrp4 gene, a Nato3 gene, an Msx1 gene, an Msx2 gene and aMash1 gene.

The detection of the dopaminergic neuron proliferative progenitor cellmarker gene other than the 187A5 gene is not particularly limited aslong as using a method by which the expression of the known gene can bedetected, and, for example, includes the hybridization method and thenucleic acid amplification method.

“The dopaminergic neuron proliferative progenitor cell marker proteinother than the 187A5 protein” includes a dopaminergic neuronproliferative progenitor cell marker protein other than the 187A5protein which is expressed in the mesencephalon most ventral ventricularzone (VZ region), and, preferably, includes a protein detected only indopaminergic neuron proliferative progenitor cells.

Such a protein includes proteins of an Lrp4 gene, a Nato3 gene, an Msx1gene, an Msx2 gene and a Mash1 gene.

The detection of the dopaminergic neuron proliferative progenitor cellmarker protein other than the 187A5 protein is not particularly limitedas long as using a method by which the expression of the known proteincan be detected, and, for example, includes the antigen-antibodyreaction method.

“The postmitotic dopaminergic neuron precursor cell marker gene or theprotein thereof” includes a gene expressed in the mesencephalon mostventral mantle layer (ML region) or a protein thereof, and includes aNurr1 gene, an En1 gene, an En2 gene, a Ptx3 gene and a TH gene.Moreover, the marker gene or the protein thereof includes a geneexpressed in the mesencephalon most ventral ventricular zone (VZ region)or a protein thereof, and includes a 65B13 gene.

The detection of the postmitotic dopaminergic neuron precursor cellmarker gene or the protein thereof is not particularly limited as longas using a method by which the expression of the known gene or theprotein thereof can be detected, and, for example, includes thehybridization method, the nucleic acid amplification method and theantigen-antibody reaction method.

The present invention provides the method for screening for an effectivesubstance for inducing differentiation into a dopaminergic neuronprogenitor cell, further comprising the following step of:

(iii-3) transforming the cell that has been contacted with the substanceto be tested, with a vector comprising a gene construct in which apromoter of the 187A5 gene is operably linked to a marker gene, anddetecting expression of the marker gene in the cell.

When the expression of the 187A5 gene, or the protein thereof isdetected in step (ii), and the expression of the marker gene is detectedin step (iii-3), the substance can be determined as the effectivesubstance for inducing differentiation into a dopaminergic neuronprogenitor cell, and preferably a dopaminergic neuron proliferativeprogenitor cell, with high accuracy.

Step (iii-3) may be performed after step (i) and may be performed beforeor after step (ii). Furthermore, step (iii-3) may be performed afterstep (iii-1) or (iii-2).

[Production Method]

The detection method according to the present invention can detect orselect dopaminergic neuron progenitor cells. The dopaminergic neuronprogenitor cells can be used in the treatment of the Parkinson'sdisease. Therefore, the dopaminergic neuron progenitor cells for use inthe treatment of the Parkinson's disease can be produced fromdopaminergic neuron progenitor cells detected or selected by usingexpression of a 187A5 gene, or a protein as an index.

The dopaminergic neuron progenitor cells used herein are preferablydopaminergic neuron proliferative progenitor cells.

The present invention provides a method for producing a dopaminergicneuron progenitor cell, comprising the following steps of:

(i) obtaining cells that can contain a dopaminergic neuron progenitorcell;(ii) detecting or selecting the dopaminergic neuron progenitor cell byusing the detection method according to the present invention; and(iii) culturing the cell obtained in step (ii).

The present invention provides a therapeutic agent for the Parkinson'sdisease, comprising dopaminergic neuron progenitor cells, and preferablydopaminergic neuron proliferative progenitor cells detected or selectedby the detection method according to the present invention.

The present invention provides use of dopaminergic neuron progenitorcells, and preferably dopaminergic neuron proliferative progenitor cellsdetected or selected by the detection method according to the presentinvention, for the production of a drug for use in the treatment of theParkinson's disease.

The present invention provides a method for treating the Parkinson'sdisease, comprising transplanting dopaminergic neuron progenitor cells,and preferably dopaminergic neuron proliferative progenitor cellsdetected or selected by the detection method according to the presentinvention, into the brain of a mammal including a human.

In the present specification, the “detection” also includes“discrimination”. Moreover, the “detection” includes not only the casethat cells as an object are discriminated as being cells of a particularkind but also the case that cells as an object are discriminated as notbeing cells of a particular kind.

EXAMPLES Example 1 Isolation and Sequence Analysis of DopaminergicNeuron Progenitor Cell-Selective Gene

An Lrp4 gene has been identified as a cell surface marker for separatingdopaminergic neuron proliferative progenitor cells (WO 2004/065599). Byusing an anti-Lrp4 antibody, it becomes possible to separatedopaminergic neuron proliferative progenitor cells derived from EScells. Thus, hereinafter, the isolation and sequence analysis of a geneselective for dopaminergic neuron proliferative progenitor cells will bedescribed.

(1) Isolation of Lrp4-Positive Cell

First, the mesencephalon and metencephalon ventral regions of a 13.5-dayrat embryo were dispersed by using the accumax (MS Techno Systems), andthen, without being subjected to fixation and permeabilizationtreatments, the cells were stained for 30 minutes at 4° C. by using ananti-Lrp4 monoclonal antibody (obtained from hybridomas (Deposition No.FERM BP-10315 and No. FERM BP-10316), diluted to 1/10, 1% fetal bovineserum (JRH), 5% fetal rat serum (JRH), 1 mM EDTA (Invitrogen)/PBS(Sigma)). Then, by using an FACS buffer (PBS+1% fetal bovine serum(JRH)+1 mM EDTA), rinsing was performed for 3 minutes at 4° C. threetimes, and the cells were stained for 20 minutes at 4° C. by using aPE-labeled anti-hamster IgG antibody (Becton Dickinson, 8 μg/ml, 1%fetal bovine serum, 5% fetal rat serum, 1 mM EDTA/PBS). Then, rinsingwas performed in the same manner. After the staining, Lrp4-positivecells were separated by a cell sorter (FACS vantage SE, BectonDickinson) (FIG. 2). The total RNA was prepared from the cellsimmediately after the separation by using the RNeasy mini kit (Qiagen),and double-strand cDNA was synthesized by using the cDNA synthesis kit(TAKARA). Next, the synthesized cDNA was digested with the restrictionenzyme RsaI (TAKARA), and then, ad2 was added thereto. The cDNA wasamplified by PCR using ad2S as a primer.

The amplification was carried out under the conditions that incubationwas performed for 5 minutes at 72° C., then, reactions for 30 seconds at94° C., for 30 seconds at 65° C. and for 2 minutes at 72° C. wereperformed at 20 cycles, and finally, incubation was performed for 2minutes at 72° C.

(SEQ ID NO: 29) ad2S: CAGCTCCACAACCTACATCATTCCGT (SEQ ID NO: 30) ad2A:ACGGAATGATGT

PCR was performed by using a reaction solution with the followingcomposition.

10xExTaq 5 μl 2.5 mM dNTP 4 μl ExTaq 0.25 μl   100 μM primer 0.5 μl  cDNA 2 μl Distilled water 38.25 μl   

Next, by using the cDNAs corresponding to the amplified cDNA of 4 ng,0.4 ng and 0.04 ng as templates, PCR was performed in the followingreaction system.

10xExTaq 1 μl 2.5 mM dNTP 0.8 μl ExTaq 0.05 μl 100 μM primer 0.1 μl foreach cDNA 1 μl Distilled water 6.95 μl

After Incubation for 2 minutes at 94° C., the amplification reactionswere performed for 30 seconds at 94° C., for 30 seconds at 65° C. andfor 2 minutes at 72° C., and finally, incubation was performed for 2minutes at 72° C. The amplifications of PCR were performed at 26 cycles.

The following primers were used in the PCR.

(SEQ ID NO: 31) Lrp4: TAGTCTACCACTGCTCGACTGTAACG (SEQ ID NO: 32)CAGAGTGAACCCAGTGGACATATCTG (SEQ ID NO: 33) Lmx1a:TGGTTCAGGTGTGGTTCCAGAACCAG (SEQ ID NO: 34) GAGTTGTAGACGCTCTGTTCAATGGC

As a result, the Lrp4 gene is expressed at the approximately equal levelin any of the mesencephalon and metencephalon Lrp4-positive cells.However, it was confirmed that an Lmx1a gene (WO 2005/052190), which isa marker gene of dopaminergic neurons and dopaminergic neuron progenitorcells, is strongly expressed only in the mesencephalon Lrp4-positivecells (FIG. 3). Therefore, it is thought that the mesencephalonLrp4-positive cells contain the dopaminergic neuron proliferativeprogenitor cells, but the metencephalon Lrp4-positive cells do notcontain the dopaminergic neuron proliferative progenitor cells.

Thus, next, by using this sample, a gene specific for the Lrp4-positivecells in the mesencephalon was searched by a subtraction (N-RDA) method(described in WO 2004/065599). As a result, one (187A5) of the isolatedcDNA fragments was a fragment encoding a functionally unknown gene.Next, expression of this gene was confirmed by the above-describedRT-PCR method using the following primers.

(SEQ ID NO: 35) 187A5: ACCAGGAAGGACAATGCCATTCGTCC (SEQ ID NO: 36)CCTTCTTCACCTTGGCTCTTAGGATG

As a result, it was confirmed that the 187A5 gene is specificallyexpressed in the Lrp4-positive cells in the mesencephalon in the samemanner as the Lmx1a gene (FIG. 3).

(2) Sequence Analysis

As a result of database search, rat and mouse cDNA sequences that arethought to be the full length of this gene were obtained (for example,Sequence Number: Mouse 187A5 AK028289, Mouse 187A5 AK157823 (frameshift), Mouse 187A5 AK028541, Mouse 187A5 XM_(—)485684 (alternative),Mouse 187A5 AK163356 (frame shift), Rat 187A5 XM_(—)344107 (predicted)).A partial sequence (SEQ ID NO: 1) that is thought to be a humanhomologous gene was also obtained, but the full length could not beobtained. Thus, homology search was performed with respect to the humangenomic sequence, and a human cDNA sequence was predicted. However, forthe neighborhood of the 5′ end, a region having high homology could notbe found. Therefore, sequence determination was performed by using a 5′RACE method.

From 1 μg of human embryonic brain mRNA (Clontech), cDNA was amplifiedby using the 5′ RACE core kit (TAKARA), and self-ligation was performed.By using the following primers, the cDNA 5′ end was amplified. Theobtained fragments were cloned into pCRII (Invitrogen), and sequencedetermination was performed.

(SEQ ID NO: 37) RT Reaction: CATCCCAGTCTC (SEQ ID NO: 38) Primary PCR:TGGAGAAGGTTGTGCCTCTGGACTTG (SEQ ID NO: 39) CTGGTTGGCTTCCTTGAGGAAGAAGG(SEQ ID NO: 40) Secondary PCR: TCCTGCGGGACAAAGTCTACCTGAGC (SEQ ID NO:41) CTGAGGATGTGGTAGCTCACAGGTAG

The PCR reaction was performed with the following composition.

10xExTaq 5 μl 2.5 mM dNTP 4 μl ExTaq 0.25 μl 100 μM primer 0.5 μl foreach Template 1 μl DMSO 1.5 μl Distilled water 37.25 μl

After incubation for 2 minutes at 94° C., the amplification reactionswere performed for 30 seconds at 94° C., for 30 seconds at 65° C. andfor 2 minutes at 72° C., and finally, incubation was performed for 2minutes at 72° C. The amplifications of PCR were performed at 35 cyclesfor primary PCR, and secondary PCR was performed by using the primaryPCR products diluted 10-fold as templates and performing amplificationsat 20 cycles.

Next, in order to confirm that the predicted sequence is correct, eachof three divided regions thereof was amplified by RT-PCR. The PCRproducts were cloned into pCRII (Invitrogen), and sequence determinationwas performed.

From 0.5 μg of human embryonic brain mRNA (Clontech), cDNA was amplifiedby using the RNA PCR kit (TAKARA). By using the cDNA as a template, PCRwas performed.

The neighborhood of the 5′ end was amplified by using the followingprimers.

(SEQ ID NO: 42) Human 187A5 F4: GAGGTCGACGCCACCATGCGCTCCGAGGGTGCGG CCCCC(SEQ ID NO: 43) Human 187a5 R1: GGGTCCATAGCTGGCATTGAGCACTG

The PCR reaction was performed with the following composition.

10xLATaq 5 μl MgCl₂ 5 μl 2.5 mM dNTP 8 μl LATaq 0.5 μl 100 μM primer 0.5μl for each cDNA 1 μl DMSO 1.5 μl Distilled water 28 μl

After incubation for 2 minutes at 94° C., the amplification reactionsfor 30 seconds at 94° C., for 30 seconds at 65° C. and for 3.5 minutesat 72° C. were performed at 35 cycles, and finally, incubation wasperformed for 2 minutes at 72° C.

The remaining regions were amplified by using the following primers F12and R5 as well as F13 and R4 in combination.

(SEQ ID NO: 44) Human 187A5 F12: CTACCTGTGAGCTACCACATCCTCAG (SEQ ID NO:45) Human 187A5 R5: TTCTCTGCCAGGATGGAGTCAGACAG (SEQ ID NO: 46) Human187A5 F13: ACTGGCAGTTCGACATCACTCACCTG (SEQ ID NO: 47) Human 187A5 R4:GAGGAATTCCAGTACAAGGAAGGCATCTGGGC AGG

As a result of sequence determination, a protein encoding this humangene exhibited a high homology to the mouse 187A5 protein over the wholeregion and had 77% amino acid identity and 87% amino acid homology.Therefore, this gene is thought to be a human 187A5 homologous gene (SEQID NO: 1).

Example 2 Expression Analysis by In Situ Hybridization of 187A5 Gene

In order to investigate the expression pattern of the 187A5 gene indetail in the cells of dopaminergic neuron lineage, expression analysisof mRNAs of 187A5 and Lrp4 was performed by in situ hybridizationaccording to the following protocol.

First, a DIG-probe was produced by the following method.

From a 12.5-day mouse (obtained from SLC) embryo, the mesencephalonmetencephalon region was cut out. The total RNA was prepared by usingthe RNeasy mini kit (Qiagen), and double-strand cDNA was synthesized byusing the cDNA synthesis kit (TAKARA). Next, by using the synthesizedcDNA as a template, cDNAs of 187A5 and Lrp4 were amplified in thefollowing reaction system.

10xExTaq 5 μl 2.5 mM dNTP 4 μl ExTaq 0.25 μl 100 μM primer 0.5 μl foreach cDNA 1 μl DMSO 1.5 μl Distilled water 37.25 μl

The amplification was carried out under the conditions that incubationwas performed for 5 minutes at 94° C., then reactions for 30 seconds at94° C., for 30 seconds at 65° C. and for 2 minutes at 72° C. wereperformed at 35 cycles, and finally, incubation was performed for 2minutes at 72° C.

The following primers were used in the PCR.

(SEQ ID NO: 48) 187A5: AGCTGAGCCACCTTCTCAGTCCAGAC (SEQ ID NO: 49)CCACGTCCAGGTCTTGACAAACCCAC (SEQ ID NO: 50) Lrp4:GACAGTGAACCTTTGGTCACTGATGG (SEQ ID NO: 51) GCCTTCCTGTCCTGGGATCAGCTTGG

The amplified cDNA fragments were cloned into pCRII (Invitrogen) andused as templates, and thereby, DIG-probes were synthesized in thefollowing reaction system (all of the reagents were purchased fromRoche).

RNA Polymerase Buffer 2 μl NTP Labeling Mix 2 μl RNase Inhibitor 1 μlRNA polymerase (T7 or SP6) 2 μl Template DNA 1 μg Distilled water Total20 μl

After 2 hours at 37° C., DNaseI (Roche) treatment was performed for 15minutes at 37° C., and the DIG-RNA probe was collected by ethanolprecipitation.

Next, a 12.5-day mouse embryo was excised and fixed for 2 hours at 4° C.by using 4% PFA (WAKO)/PBS. Then, the solution was replaced at 4° C.overnight by 20% sucrose (WAKO)/PBS, and then, the embryo was embeddedwith OCT (Sakura Seiki Co., Ltd.). Sections of 12 μm thickness wereprepared, dried on slide glasses, and then fixed again for 30 minutes atroom temperature by using 4% PFA. After rinsing with PBS, hybridization(1 μg/ml DIG-RNA probe, 50% formamide (Nacalai Tesque, Inc.), 5×SSC, 1%SDS, 50 μg/ml yeast RNA (Sigma), 50 μg/ml heparin) was performed for 40hours at 68° C. Then, rinsing (50% formamide, 5×SSC, 1% SDS) wasperformed at 68° C., and further rinsing (50% formamide, 5×SSC) wasperformed at 68° C. After rinsing with 1×TBST at room temperature,blocking (blocking agent: Roche) was performed. An alkalinephosphatase-labeled anti-DIG antibody (DAKO) was reacted therewith at 4°C. overnight, and after rinsing (1×TBST, 2 mM levamisole), NBT/BCIP(DAKO) was used as the substrate for coloring.

As a result, in the 12.5-day mouse embryo which is in the period ofgenerating dopaminergic neurons, it became revealed that mRNA of 187A5is selectively expressed in the mesencephalon most ventral ventricularzone (ventricular zone: VZ) in which the Lrp4-positive dopaminergicneuron progenitor cells exist and the mesencephalon most dorsal roofplate region (FIG. 4). On the other hand, the expression was notrecognized in the metencephalon ventral region. Therefore, it wasconfirmed that mRNA of 187A5 is not expressed in the metencephalon floorplate cells positive for Lrp4.

From the above-described results, it became revealed that mRNA of 187A5is selectively expressed in the dopaminergic neuron proliferativeprogenitor cells. Cells simultaneously expressing both of the Lrp4 and187A5 genes are limited to the dopaminergic neuron proliferativeprogenitor cells that exist in the mesencephalon most ventralventricular zone. Therefore, it is thought that the dopaminergic neuronproliferative progenitor cells can be discriminated with higher accuracyby using these markers in combination.

Example 3 Expression of 187A5 Gene in Dopaminergic Neurons Induced toDifferentiate from ES Cells

Whether or not the 187A5 gene is expressed when ES cells are induced todifferentiate into dopaminergic neurons in vitro was studied.

First, according to the SDIA method (Kawasaki et al. Neuron. 2000October; 28 (1): 31-40), ES cells (mouse-derived CCE strain providedfrom Mr. Nishikawa in Riken CDB, Kawasaki et al. Neuron. 2000 28 (1):31-40.) were induced to differentiate into dopaminergic neurons.Lrp4-positive and Lrp4-negative cells were separated from the cells inthe sixth day after the induction (Example 5 of WO 2004/065599), and thetotal RNA was prepared from the cells immediately after the separation.By using this total RNA as a template, cDNA was synthesized andamplified.

Moreover, according to the 5-stage method (Lee et al. (2000) Nat.Biotech. 18: 675-679, mouse dopaminergic neuron differentiation kit (R &D Systems)), ES cells (CCE) were induced to differentiate intodopaminergic neurons. Lrp4-positive and Lrp4-negative cells wereseparated from the cells in the seventh day of stage 4 (Example 8 of WO2004/065599), and the total RNA was prepared from the cells immediatelyafter the separation. By using this total RNA as a template, cDNA wassynthesized and amplified.

Next, by using the cDNAs corresponding to the amplified cDNA of 4 ng,0.4 ng and 0.04 ng as templates, PCR was performed in the followingreaction system.

10xExTaq 1 μl 2.5 mM dNTP 0.8 μl ExTaq 0.05 μl 100 μM primer 0.1 μl foreach cDNA 1 μl DMSO 0.3 μl Distilled water 6.65 μl

After incubation for 2 minutes at 94° C., the amplification reactionswere performed for 30 seconds at 94° C., for 30 seconds at 65° C. andfor 2 minutes at 72° C., and finally, incubation was performed for 2minutes at 72° C. The amplifications of PCR were performed at 26 cycles.

The following primers were used in the PCR.

(SEQ ID NO: 33) Lmx1a: TGGTTCAGGTGTGGTTCCAGAACCAG (SEQ ID NO: 52)TCTGAGGTTGCCAGGAAGCAGTCTCC

In addition, for Lrp4 and 187A5, the primers of Example 1 were used.

As a result, it was confirmed that the 187A5 gene is expressed in thedifferentiation induction by any of the methods, and strongly expressed,particularly, in the Lrp4-positive cells (FIGS. 5 and 6). Therefore, itbecame revealed that mRNA of 187A5 is expressed in the dopaminergicneuron progenitor cells not only in the cells derived from the mouse andrat embryonic mesencephalons but also in the cells induced todifferentiate by any of the SDIA method and the 5-stage method.Specifically, it became revealed that the 187A5 gene serves as a usefulmarker for discriminating not only the dopaminergic neuron progenitorcells derived from the embryonic mesencephalon but also the dopaminergicneuron progenitor cells induced to differentiate from ES cells in vitro.

Example 4 Expression of 187A5 Protein on Cell Surface

In the 187A5 protein, a sequence that is thought to be a transmembraneregion exists at one site. If the 187A5 protein is expressed on the cellsurface, 187A5-positive live cells can be separated by flow cytometryusing an antibody capable of binding to the 187A5 protein and areexpected to be useful in preparation of a transplant material for theParkinson's disease or the like. Therefore, the intracellularlocalization of the 187A5 protein was studied.

(1) Analysis of Signal Sequence

In the case of a type I transmembrane protein, a signal sequencegenerally exists in the neighborhood of the N-terminal and is cleavedoff immediately after the signal sequence, and thereby, the protein canbe expressed on the membrane. As a result of computer search (PSORT II,http://psort.ims.u-tokyo.ac.jp/form2.html), a sequence that is predictedto be a signal sequence was not found in the mouse 187A5 gene. On theother hand, a signal sequence-like sequence existed in the neighborhoodof the N-terminal of the human 187A5 gene. Therefore, whether afunctional signal sequence exists in the 187A5 gene was studied.

A construct in which a region encoding the amino acids from theN-terminal to amino acid 45 in mouse cDNA was linked to signalsequence-deficient secreted alkaline phosphatase cDNA was prepared andtransfected into 293E cells. A culture supernatant in the fourth day ofculture was collected, and alkaline phosphatase activity was measured byusing the Aurora kit (ICN) (FIG. 7).

As a result, when signal sequence-deficient secreted alkalinephosphatase (control) is expressed, this protein is not secreted.Therefore, alkaline phosphatase activity is not recognized in thesupernatant. By contrast, in the case of the fusion protein in which theN-terminal sequence was linked, strong activity was recognized in thesupernatant. Therefore, it became revealed that the fusion protein isefficiently secreted by the N-terminal sequence of 187A5 (FIG. 8).Therefore, it became revealed that a functional signal sequence existsin the neighborhood of the N-terminal of 187A5. This indicates that the187A5 protein is a type I single transmembrane molecule.

(2) Expression of 187A5 on Cell Surface (Biotinylation Method)

In order to confirm whether or not the 187A5 protein is expressed on thecell surface, whether the 187A5 protein is biotinylated when onlyproteins on the cell surface are biotin-labeled was studied.

A construct in which an HA tag was added to the C-terminal of 187A5 wastransfected into NS20Y cells. After 2 days, the cells were rinsed withcold PBS twice, and then, 5 ml of 0.5 mg/ml EZ-link Sulfo-NHS-SS-Biotin(PIERCE) (dissolved in PBS+1 mM CaCl₂, 0.5 mM MgCl₂) was added thereto.The reaction was performed for 30 minutes at room temperature. Afterrinsing with cold PBS twice, the cells were collected, then suspended in600 μl of a dissolution buffer (1% SDS, 10 mM Tris-Cl, 100 mM NaCl, 1 mMEDTA), and subjected to ultrasonication. After centrifugation for 3minutes at 14000 rpm, the supernatant was collected. 20 μl ofstreptavidin beads (PIERCE) was added thereto, and after rotation for 1hour at room temperature, rinsing was performed with a dissolutionbuffer twice. To the beads, 75 μl of SDS-PAGE sample buffer was added,and after 3 minutes at 100° C., the bound proteins were collected bycentrifugation. The 187A5 protein was detected by western blotting usingan anti-HA antibody (Roche).

As a result, it became revealed that the 187A5 protein is biotinylatedwith high efficiency (FIG. 9). Therefore, it is thought that the 187A5protein is expressed on the cell surface.

(3) Expression of 187A5 on Cell Surface (FACS Analysis)

Whether the 187A5 protein can be detected by a FACS method was studied.A construct in which cDNA encoding the C-terminal side from thepredicted cleavage site (amino acid 39) of 187A5 was linked immediatelyafter a signal sequence of Preprotrypsin and a sequence encoding a FLAGtag was prepared. By expressing this construct, 187A5 in which the FLAGtag is added to the N-terminal can be expressed after the cleavage ofthe signal sequence. This construct was stably introduced into B300.19cells through retrovirus vectors. The parent cells and transformantswere rinsed with a FACS buffer (PBS+1% fetal bovine serum (JRH)+1 mMEDTA). Then, reaction with 10 μg/ml anti-FLAG antibody (SIGMA) wasperformed for 30 minutes on ice, and rinsing was performed with a FACSbuffer. Sequentially, reaction with a PE-labeled anti-mouse IgG antibody(Jackson) (diluted to 1/200) was performed for 30 minutes on ice, andrinsing was performed with a FACS buffer. After staining, analysis wasperformed by flow cytometry (FACS calibur, Becton Dickinson).

As a result, unlike the parent strains, a population that stronglyreacts with the FLAG antibody was detected in the stable transformants(FIG. 10). Therefore, it became revealed that 187A5 is expressed on thecell surface in a direction wherein the N-terminal side thereof can belocated in the extracellular space, and can be detected by FACS using anantibody. Specifically, it is thought that 187A5 is useful as a markerfor separating dopaminergic neuron progenitor cells as live cells.

Example 5 Expression Analysis of 187A5 Protein

By using a gene sequence encoding the extracellular region in the 187A5gene, an anti-187A5 antibody was produced according to the followingprotocol, and expression analysis was performed by immunohistologicstaining.

First, a gene sequence encoding the extracellular region (amino acids 1to 919 of SEQ ID NO: 15) in the mouse 187A5 gene was gene-transfectedinto 293E cells, and the extracellular region of the 187A5 protein wasexpressed and collected. A rat was immunized with the collected protein,and then, lymphocytic cells were extracted and cell-fused with myelomacells. From the fused cell population, a clone capable of reacting with187A5 was selected. An anti-187A5 monoclonal antibody was purified froma culture supernatant of this clone. Next, an 11.5-day mouse embryo wasfixed for 2 hours at 4° C. by using 4% PFA/PBS (−). Then, the solutionwas replaced at 4° C. overnight by 20% sucrose/PBS (−), and then, theembryo was embedded with OCT. Sections of 12 μm thickness were prepared,mounted on slide glasses, then dried for 30 minutes at room temperature,and moistened again with PBS (−). Then, blocking (25% Blockace(Dainippon Sumitomo Pharma Co., Ltd.)) was performed for 30 minutes atroom temperature. The prepared anti-187A5 monoclonal antibody (culturesupernatant diluted 2-fold, 2.5% Blockace/PBS) was reacted therewith for2.5 hours at room temperature, and then, rinsing was performed for 10minutes at room temperature four times by using 0.01% Triton X-100/PBS(−). A Cy3-labeled anti-rat IgG antibody (Jackson, 10 μg/ml, 2.5%Blockace/PBS) was reacted therewith for 1 hour at room temperature, andrinsing was performed in the same manner. Then, rinsing with PBS (−) wasperformed for 5 minutes at room temperature, and sealing was performed.

As a result of expression analysis by immunohistologic staining usingthe prepared anti-187A5 monoclonal antibody, as with the results ofExample 2, the existence of the 187A5 protein was recognized in themesencephalon ventral region of E11.5 which is in the period ofgenerating dopaminergic neurons, and was not recognized in themetencephalon ventral region in which dopaminergic neurons are notgenerated (FIG. 11).

From these results, it was confirmed that the 187A5 protein exists indopaminergic neuron progenitor cells.

Example 6 Detection of Cell in which 187A5 Protein Exists

By using the anti-187A5 monoclonal antibody prepared in Example 5, cellsin which the 187A5 protein exists were detected by flow cytometry.

First, the mesencephalon and metencephalon ventral regions of a mouseE12.5 embryo were dispersed by using the cell dissociation buffer(Invitrogen), and then, without being subjected to fixation andpermeabilization treatments, the cells were stained for 20 minutes at 4°C. by using the anti-187A5 monoclonal antibody (purified antibodydiluted to 1/10, 1% fetal bovine serum, 1 mM EDTA/PBS) and an anti-Lrp4antibody (culture supernatant diluted to 1/2, 1% fetal bovine serum, 1mM EDTA/PBS). Then, by using 1% fetal bovine serum and 1 mM EDTA/PBS-,rinsing was performed for 3 minutes at 4° C. three times. The cells werestained for 20 minutes at 4° C. by using a biotin-labeled anti-Armenianhamster IgG antibody (Jackson, 10 μg/ml, 1% fetal bovine serum, 1 mMEDTA/PBS), and rinsing was performed in the same manner. Then, the cellswere stained for 20 minutes at 4° C. by using APC-labeled streptavidin(Pharmingen, 8 μg/ml, 1% fetal bovine serum, 1 mM EDTA/PBS) and aPE-labeled anti-rat IgG antibody (Jackson, 20 μg/ml, 1% fetal bovineserum, 1 mM EDTA/PBS), and rinsing was performed in the same manner.After the staining, detection was performed by using a flow cytometer.

As a result of flow cytometry by using the prepared anti-187A5monoclonal antibody, a cell population in which the 187A5 protein existswas detected (FIG. 12). Here, the cells in which the 187A5 proteinexists can be detected without being subjected to fixation andpermeabilization treatments. Therefore, it was suggested that the cellsin which the 187A5 protein exists can be separated as live cells byusing a flow cytometer equipped with a cell sorter. Moreover, it wasconfirmed that the 187A5 protein exists in all of the mesencephalonLrp4-positive cells, namely, the dopaminergic neuron progenitor cells.On the other hand, it was confirmed that the 187A5 protein does notexist in the metencephalon Lrp4-positive cells which do not containdopaminergic neuron progenitor cells (FIG. 12).

From these results, it was shown that the 187A5 antibody is useful forseparating dopaminergic neuron progenitor cells.

Example 7 Expression of 187A5 Protein in Dopaminergic Neurons Induced toDifferentiate from ES Cells

The group of the cells containing the dopaminergic neuron progenitorcells induced to differentiate from ES cells in vitro by the SDIA methodwas dispersed by using the cell dissociation buffer (Invitrogen), andthen, without being subjected to fixation and permeabilizationtreatments, the cells were stained for 20 minutes at 4° C. by using theanti-187A5 monoclonal antibody (purified antibody diluted to 1/10, 10%knockout serum replacement, 1% fetal bovine serum, 1 mM EDTA/SDIAdifferentiation medium) prepared in Example 5 and an anti-Lrp4 antibody(culture supernatant diluted to 1/2, 10% knockout serum replacement, 1%fetal bovine serum, 1 mM EDTA/SDIA differentiation medium). Then, byusing 10% knockout serum replacement, 1% fetal bovine serum and 1 mMEDTA/SDIA differentiation medium, rinsing was performed for 3 minutes at4° C. three times. The cells were stained for 20 minutes at 4° C. byusing a biotin-labeled anti-Armenian hamster IgG antibody (Jackson, 10μg/ml, 10% knockout serum replacement, 1% fetal bovine serum, 1 mMEDTA/SDIA differentiation medium), and rinsing was performed in the samemanner. Then, the cells were stained for 20 minutes at 4° C. by usingAPC-labeled streptavidin (Pharmingen, 8 μg/ml, 10% knockout serumreplacement, 1% fetal bovine serum, 1 mM EDTA/SDIA differentiationmedium) and a PE-labeled anti-rat IgG antibody (Jackson, 20 μg/ml, 10%knockout serum replacement, 1% fetal bovine serum, 1 mM EDTA/SDIAdifferentiation medium), and rinsing was performed in the same manner.After the staining, 187A5- and Lrp4-expressing cells were detected byusing a flow cytometer.

As a result of flow cytometry, a cell population in which the 187A5 andLrp4 proteins exist was detected in the same manner as the mouseembryonic mesencephalon (FIG. 13).

From these results, it was shown that the 187A5 antibody is also usefulfor separating dopaminergic neuron progenitor cells derived from EScells.

Example 8 Separation of Lrp4-Expressing Cell by Using Antibody

In order to confirm that the separated 187A5/Lrp4-copositive cellsdifferentiate into dopaminergic neurons, the following experiment wasperformed by using Nurr1, a postmitotic dopaminergic neuron precursorcell marker.

The cells separated after being induced to differentiate from ES cellsin vitro by the SDIA method were inoculated onto a slide glass coatedwith poly-L-ornithine (Sigma, 0.002% in PBS), laminin (Invitrogen, 2.5μg/ml in PBS) and fibronectin (Sigma, 5 μg/ml in PBS), and cultured for6 days at 37° C. in N2 (Invitrogen, 1×), B27 (Invitrogen, 1×), ascorbicacid (Sigma, 200 uM) BDNF (Invitrogen, 20 ng/ml) and 10% knockout serumreplacement (Invitrogen)/SDIA differentiation medium. The cultured cellswere fixed for 20 minutes at 4° C. by using 2% PFA/PBS, and rinsing withPBS was performed for 10 minutes at 4° C. twice. Then, permeabilizationtreatment with 0.3% Triton X-100/PBS was performed for 30 minutes atroom temperature, and blocking was performed for 20 minutes at roomtemperature with 10% normal donkey serum/Blockace. Sequentially,reaction with an anti-Nurr1 antibody (in house culture supernatantdiluted to 1/1000, 10% normal donkey serum, 2.5% Blockace, 0.1% TritonX-100/PBS) and an anti-HuC/D antibody (Molecular Probe, 1/50, 4 μg/ml,10% normal donkey serum, 2.5% Blockace, 0.1% Triton X-100/PBS) wasperformed for 1 hour at room temperature, and sequentially, the reactionwas performed overnight at 4° C. On the next day, by using 0.1% TritonX-100/PBS, rinsing was performed for 10 minutes at room temperature fourtimes. Then, reaction with an FITC-labeled anti-mouse IgG antibody and aCy3-labeled anti-rat IgG antibody (all Jackson, 3 μg/ml, 10% normaldonkey serum, 2.5% Blockace, 0.1% Triton X-100/PBS) was performed for 1hour at room temperature. Then, rinsing was performed in the samemanner, and rinsing with PBS was performed for 5 minutes at roomtemperature. After sealing, the cells were observed.

As a result of culturing the cells separated by flow cytometry for 6days in vitro, evidently many Nurr1-positive dopaminergic neurons wereinduced, compared to unseparated cells as controls (FIG. 14).

From these results, it became revealed that the 187A5/Lrp4-copositivecells were certainly progenitor cells of dopaminergic neuron lineage andcan be matured in vitro.

1. A probe or primer for use in the detection or selection of adopaminergic neuron progenitor cell, which can hybridize to a nucleotidesequence, or a complementary sequence thereto, of a polynucleotideselected from the following (i), (ii), (iii) and (iv): (i) apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; (ii)a polynucleotide encoding a protein which consists of an amino acidsequence encoded by a nucleotide sequence of SEQ ID NO: 1 in which oneor more nucleotides are inserted, substituted and/or deleted, and/or oneor more nucleotides are added to one or both of ends, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2; (iii) a polynucleotide which hybridizes understringent conditions to a polynucleotide consisting of the complementarysequence of the nucleotide sequence of SEQ ID NO: 1, and which encodes aprotein functionally equivalent to a protein consisting of the aminoacid sequence of SEQ ID NO: 2; and (iv) a polynucleotide which has 70%or more identity with a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 1, and which encodes a protein functionallyequivalent to a protein consisting of the amino acid sequence of SEQ IDNO: 2; or a polynucleotide encoding a protein selected from thefollowing (v), (vi), (vii) and (viii): (v) a protein comprising theamino acid sequence of SEQ ID NO: 2; (vi) a protein which consists of anamino acid sequence of SEQ ID NO: 2 in which one or more amino acids areinserted, substituted and/or deleted, and/or one or more amino acids areadded to one or both of ends, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; (vii) aprotein which is encoded by a polynucleotide which hybridizes understringent conditions to a polynucleotide consisting of a complementarysequence of a nucleotide sequence of a polynucleotide encoding the aminoacid sequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO:
 2. 2. The probe or primer according to claim 1,wherein the polynucleotide is derived from a human, a mouse, a rat, abovine, a dog or a chimpanzee.
 3. The probe or primer according to claim1, wherein the nucleotide sequence of the polynucleotide is selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 25 and SEQ ID NO:
 27. 4. The probe or primer according toclaim 1, wherein the nucleotide sequence of the polynucleotide is anucleotide sequence comprising a part or all of a nucleotide sequence ofnucleotides 774 to 1221 or 2403 to 2666 of SEQ ID NO:
 1. 5. The probe orprimer according to claim 1, which consists of a polynucleotidecomprising at least 10 contiguous nucleotides of the nucleotide sequenceof the polynucleotide, or the complementary sequence thereto.
 6. Theprobe or primer according to claim 1, which has at least 25 base length.7. The probe or primer according to claim 1, wherein the dopaminergicneuron progenitor cell is a dopaminergic neuron proliferative progenitorcell.
 8. A primer set consisting of two or more primers according toclaim
 1. 9. An antibody capable of binding to a protein, or a partthereof, wherein the protein is selected from the following (v), (vi),(vii) and (viii): (v) a protein comprising the amino acid sequence ofSEQ ID NO: 2; (vi) a protein which consists of an amino acid sequence ofSEQ ID NO: 2 in which one or more amino acids are inserted, substitutedand/or deleted, and/or one or more amino acids are added to one or bothof ends, and which is functionally equivalent to a protein consisting ofthe amino acid sequence of SEQ ID NO: 2; (vii) a protein which isencoded by a polynucleotide which hybridizes under stringent conditionsto a polynucleotide consisting of a complementary sequence of anucleotide sequence of a polynucleotide encoding the amino acid sequenceof SEQ ID NO: 2, and which is functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; and (viii) aprotein which consists of an amino acid sequence having 70% or moreidentity with the amino acid sequence of SEQ ID NO: 2, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO:
 2. 10. The antibody according to claim 9, whereinthe protein, or a part thereof is a protein comprising a polypeptideconsisting of at least 5 amino acid residues or all of an amino acidsequence of amino acids 248 to 397 or 792 to 877 of SEQ ID NO:
 2. 11.The antibody according to claim 9, wherein the protein, or a partthereof is a polypeptide region expressed in the extracellular space.12. The antibody according to claim 11, wherein the protein, or a partthereof comprises a polypeptide consisting of at least 5 amino acidresidues or all of an amino acid sequence of amino acids 28 to 927 ofSEQ ID NO: 2, amino acids 16 to 1267 of SEQ ID NO: 4, amino acids 1 to550 of SEQ ID NO: 6, amino acids 1 to 542 of SEQ ID NO: 8, amino acids 1to 418 of SEQ ID NO: 10, amino acids 76 to 964 of SEQ ID NO: 12, aminoacids 40 to 928 of SEQ ID NO: 15, amino acids 1 to 540 of SEQ ID NO: 17,amino acids 40 to 1106 of SEQ ID NO: 19, amino acids 24 to 1524 of SEQID NO: 22, amino acids 43 to 1018 of SEQ ID NO: 24, amino acids 43 to908 of SEQ ID NO: 26 or amino acids 1 to 866 of SEQ ID NO:
 28. 13. Theantibody according to claim 9, wherein the protein, or a part thereof isa polypeptide consisting of at least 6 amino acid residues.
 14. Theantibody according to claim 9, wherein the dopaminergic neuronprogenitor cell is a dopaminergic neuron proliferative progenitor cell.15. A method for detecting or selecting a dopaminergic neuron progenitorcell, comprising the step of detecting expression of a polynucleotideselected from the following (i), (iii) and (iv): (i) a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 1; (ii) apolynucleotide encoding a protein which consists of an amino acidsequence encoded by a nucleotide sequence of SEQ ID NO: 1 in which oneor more nucleotides are inserted, substituted and/or deleted, and/or oneor more nucleotides are added to one or both of ends, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO: 2; (iii) a polynucleotide which hybridizes understringent conditions to a polynucleotide consisting of the complementarysequence of nucleotide which encodes a protein functionally equivalentto a protein consisting of the amino acid sequence of SEQ ID NO: 2; and(iv) a polynucleotide which has 70% or more identity with apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; or the step ofdetecting a protein selected from the following (v), (vi), (vii) and(viii): (v) a protein comprising the amino acid sequence of SEQ ID NO:2; (vi) a protein which consists of an amino acid sequence of SEQ ID NO:2 in which one or more amino acids are inserted, substituted and/ordeleted, and/or one or more amino acids are added to one or both ofends, and which is functionally equivalent to a protein consisting ofthe amino acid sequence of SEQ ID NO: 2; (vii) a protein which isencoded by a polynucleotide which hybridizes under stringent conditionsto a polynucleotide consisting of a complementary sequence of anucleotide sequence of a polynucleotide encoding the amino acid sequenceof SEQ ID NO: 2, and which is functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; and (viii) aprotein which consists of an amino acid sequence having 70% or moreidentity with the amino acid sequence of SEQ ID NO: 2 and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO:
 2. 16. The method according to claim 15, whereinthe dopaminergic neuron progenitor cell is a dopaminergic neuronproliferative progenitor cell.
 17. The method according to claim 15,wherein the step of detecting expression of a polynucleotide comprisesthe following steps of: (a) contacting a cell sample to be tested, witha probe or a primer set according to claim 1; and (b) detecting thepresence or absence of reactivity.
 18. The method according to claim 15,wherein the step of detecting expression of the polynucleotide comprisesthe following steps of: (a-1) contacting the polynucleotide derived froma cell sample to be tested with a probe claim 1; and (b-1) detecting ahybridization complex.
 19. The method according to claim 18, wherein instep (a-1), mRNA prepared from the cell sample to be tested or acomplementary DNA transcribed from the mRNA is contacted with the probe.20. The method according to claim 15, wherein the step of detectingexpression of the polynucleotide comprises the steps of: (a-2)performing a nucleic acid amplification method by using a polynucleotidederived from a cell sample to be tested as a template and a primer setaccording to claim 8; and (b-2) detecting a formed amplificationproduct.
 21. The method according to claim 20, wherein in step (a-2),mRNA prepared from the cell sample to be tested or a complementary DNAtranscribed from the mRNA is used as a template.
 22. The methodaccording to claim 15, wherein the step of detecting the protein furthercomprises the following steps of: (c) contacting a cell sample to betested, with an antibody claim 9; and (d) detecting the presence orabsence of reactivity.
 23. The method according to claim 15, wherein thestep of detecting the protein comprises the following steps of: (c-1)contacting the protein derived from a cell sample to be tested, with anantibody claim 9; and (d-1) detecting an antigen-antibody complex. 24.The method according to claim 17, wherein the cell sample to be testedis an ES cell induced to differentiate into a dopaminergic neuronproliferative progenitor cell.
 25. The method according to claim 24,wherein the differentiation induction is carried out by an SDIA method.26. The method according to claim 17, wherein the cell sample to betested is a cell obtained from an embryonic mesencephalon ventralregion.
 27. The method according to claim 17, wherein the cell sample tobe tested comprise a cell selected from the group consisting of: a cellin which expression of a dopaminergic neuron proliferative progenitorcell marker gene other than the polynucleotide is detected, a cell inwhich a dopaminergic neuron proliferative progenitor cell marker proteinother than the protein is detected; a cell in which expression of apostmitotic dopaminergic neuron precursor cell marker gene, or a proteinthereof is not detected; a cell in which expression of a dopaminergicneuron progenitor cell marker gene other than the polynucleotide isdetected, a cell in which a dopaminergic neuron progenitor cell markerprotein other than the protein is detected; and a cell in whichexpression of a mature dopaminergic neuron cell marker gene, or aprotein thereof is not detected.
 28. The method according to claim 17,which further comprises the step of, after step (b) or step (d): (e-1)detecting expression of a dopaminergic neuron proliferative progenitorcell marker gene other than the polynucleotide, or a dopaminergic neuronproliferative progenitor cell marker protein other than the protein;(e-2) detecting expression of a postmitotic dopaminergic neuronprecursor cell marker gene, or a protein thereof; (e-3) detectingexpression of a dopaminergic neuron progenitor cell marker gene otherthan the polynucleotide, or a dopaminergic neuron progenitor cell markerprotein other than the protein; or (e-4) detecting expression of amature dopaminergic neuron cell marker gene, or a protein thereof. 29.The method according to claim 27, wherein the dopaminergic neuronproliferative progenitor cell marker gene other than the polynucleotideis a gene expressed in the mesencephalon most ventral ventricular zone.30. The method according to claim 27, wherein the dopaminergic neuronproliferative progenitor cell marker gene other than the polynucleotideis selected from the group consisting of an Lrp4 gene, an Msx1 gene, anMsx2 gene, a Nato3 gene and a Mash1 gene.
 31. The method according toclaim 27, wherein the dopaminergic neuron proliferative progenitor cellmarker protein other than the protein is a protein expressed in themesencephalon most ventral ventricular zone.
 32. The method according toclaim 27, wherein the dopaminergic neuron proliferative progenitor cellmarker protein other than the protein is a protein of a gene selectedfrom the group consisting of an Lrp4 gene, an Msx1 gene, an Msx2 gene, aNato3 gene and a Mash1 gene.
 33. The method according to claim 27,wherein the postmitotic dopaminergic neuron precursor cell marker geneis a gene expressed in the mesencephalon most ventral mantle layerregion.
 34. The method according to claim 27, wherein the postmitoticdopaminergic neuron precursor cell marker gene is selected from thegroup consisting of a Nurr1 gene, an En1 gene, an En2 gene, a Ptx3 gene,a TH gene and a 65B13 gene.
 35. The method according to claim 27,wherein the dopaminergic neuron progenitor cell marker gene other thanthe polynucleotide is a gene expressed in the mesencephalon most ventralregion.
 36. The method according to claim 27, wherein the dopaminergicneuron progenitor cell marker gene other than the polynucleotide is anLmx1a gene.
 37. The method according to claim 27, wherein thedopaminergic neuron progenitor cell marker protein other than theprotein is a protein expressed in the mesencephalon most ventral region.38. The method according to claim 27, wherein the dopaminergic neuronprogenitor cell marker protein other than the protein is a protein of anLmx1a gene.
 39. The method according to claim 27, wherein the maturedopaminergic neuron cell marker gene is a DAT gene.
 40. The methodaccording to claim 17, which further comprises the steps of transformingthe cell sample to be tested, with a vector comprising a gene constructin which a promoter of the polynucleotide is operably linked to a markergene, and detecting expression of the marker gene in the cell sample tobe tested.
 41. A kit for detecting or selecting a dopaminergic neuronprogenitor cell, comprising at least a probe or primer according toclaim 1, a primer set according to claim 8 or an antibody according toclaim
 9. 42. The kit according to claim 41, wherein the dopaminergicneuron progenitor cell is a dopaminergic neuron proliferative progenitorcell.
 43. The kit according to claim 41, which further comprises: aprobe, a primer, a primer set or an antibody, which can detectexpression of a dopaminergic neuron proliferative progenitor cell markergene other than the polynucleotide, or a dopaminergic neuronproliferative progenitor cell marker protein other than the protein; aprobe, a primer, a primer set or an antibody, which can detectexpression of a postmitotic dopaminergic neuron precursor cell markergene, or a protein thereof; a probe, a primer, a primer set or anantibody, which can detect expression of a dopaminergic neuronprogenitor cell marker gene other than the polynucleotide, or adopaminergic neuron progenitor cell marker protein other than theprotein; or a probe, a primer, a primer set or an antibody, which candetect expression of a mature dopaminergic neuron cell marker gene, or aprotein thereof.
 44. The kit according to claim 41, which furthercomprises a vector comprising a gene construct in which a promoter ofthe polynucleotide is operably linked to a marker gene.
 45. An agent fordetecting or selecting a dopaminergic neuron progenitor cell, comprisingat least a probe or primer according to claim 1, a primer set accordingto claim 8 or an antibody according to claim
 9. 46. The agent accordingto claim 45, wherein the dopaminergic neuron progenitor cell is adopaminergic neuron proliferative progenitor cell.
 47. The agentaccording to claim 45, which further comprises: a probe, a primer, aprimer set or an antibody, which can detect expression of a dopaminergicneuron proliferative progenitor cell marker gene other than thepolynucleotide, or a dopaminergic neuron proliferative progenitor cellmarker protein other than the protein; a probe, a primer, a primer setor an antibody, which can detect expression of a postmitoticdopaminergic neuron precursor cell marker gene, or a protein thereof; aprobe, a primer, a primer set or an antibody, which can detectexpression of a dopaminergic neuron progenitor cell marker gene otherthan the polynucleotide, or a dopaminergic neuron progenitor cell markerprotein other than the protein; or a probe, a primer, a primer set or anantibody, which can detect expression of a mature dopaminergic neuroncell marker gene, or a protein thereof.
 48. The agent according to claim45, which further comprises a vector comprising a gene construct inwhich a promoter of the polynucleotide is operably linked to a markergene.
 49. A method for screening for an effective substance for inducingdifferentiation into a dopaminergic neuron progenitor cell, comprisingthe following steps of: (i) contacting a cell that can differentiateinto a dopaminergic neuron progenitor cell, with a substance to betested; and (ii) detecting in the cell that has been contacted with thesubstance to be tested expression of a polynucleotide selected from thefollowing (i), (ii), (iii) and (iv): (i) a polynucleotide comprising thenucleotide sequence of SEQ ID NO: 1; (ii) a polynucleotide encoding aprotein which consists of an amino acid sequence encoded by a nucleotidesequence of SEQ ID NO: 1 in which one or more nucleotides are inserted,substituted and/or deleted, and/or one or more nucleotides are added toone or both of ends, and which is functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; (iii) apolynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of the complementary sequence of nucleotidesequence of SEQ ID NO: 1, and which encodes a protein functionallyequivalent to a protein consisting of the amino acid sequence of SEQ IDNO: 2; and (iv) a polynucleotide which has 70% or more identity with apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; or a proteinselected from the following (v), (vi), (vii) and (viii): (v) a proteincomprising the amino acid sequence of SEQ ID NO: 2; (vi) a protein whichconsists of an amino acid sequence of SEQ ID NO: 2 in which one or moreamino acids are inserted, substituted and/or deleted, and/or one or moreamino acids are added to one or both of ends, and which is functionallyequivalent to a protein consisting of the amino acid sequence of SEQ IDNO: 2; (vii) a protein which is encoded by a polynucleotide whichhybridizes under stringent conditions to a polynucleotide c the aminoacid sequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO:
 2. 50. The screening method according to claim49, wherein the dopaminergic neuron progenitor cell is a dopaminergicneuron proliferative progenitor cell.
 51. The method according to claim50, which further comprises the step of: (iii-1) detecting expression ofa dopaminergic neuron proliferative progenitor cell marker gene otherthan the polynucleotide, or a dopaminergic neuron proliferativeprogenitor cell marker protein other than the protein in the cell thathas been contacted with the substance to be tested.
 52. The methodaccording to claim 51, wherein the dopaminergic neuron proliferativeprogenitor cell marker gene other than the polynucleotide is a geneexpressed in the mesencephalon most ventral ventricular zone.
 53. Themethod according to claim 51, wherein the dopaminergic neuronproliferative progenitor cell marker gene other than the polynucleotideis selected from the group consisting of an Lrp4 gene, an Msx1 gene, anMsx2 gene, a Nato3 gene and a Mash1 gene.
 54. The method according toclaim 51, wherein the dopaminergic neuron proliferative progenitor cellmarker protein other than the protein is a protein expressed in themesencephalon most ventral ventricular zone.
 55. The method according toclaim 51, wherein the dopaminergic neuron proliferative progenitor cellmarker protein other than the protein is a protein of a gene selectedfrom the group consisting of an Lrp4 gene, an Msx1 gene, an Msx2 gene, aNato3 gene and a Mash1 gene.
 56. The method according to claim 50, whichfurther comprises the step of: (iii-2) detecting expression of apostmitotic dopaminergic neuron precursor cell marker gene, or a proteinthereof in the cell that has been contacted with the substance to betested.
 57. The method according to claim 56, wherein the postmitoticdopaminergic neuron precursor cell marker gene is a gene expressed inthe mesencephalon most ventral mantle layer region.
 58. The methodaccording to claim 56, wherein the postmitotic dopaminergic neuronprecursor cell marker gene is selected from the group consisting of aNurr1 gene, an En1 gene, an En2 gene, a Ptx3 gene, a TH gene and a 65B13gene.
 59. The method according to claim 50, which further comprises thestep of: (iii-3) transforming the cell that has been contacted with thesubstance to be tested, with a vector comprising a gene construct inwhich a promoter of the polynucleotide is operably linked to a markergene, and detecting expression of the marker gene in the cell.
 60. Amethod for producing a dopaminergic neuron progenitor cell, comprisingthe steps of: (i) obtaining cells that can contain a dopaminergic neuronprogenitor cell; (ii) detecting the dopaminergic neuron progenitor cellby using the method according to claim 15; and (iii) culturing the celldetected or selected in step (ii).
 61. The production method accordingto claim 60, wherein the dopaminergic neuron progenitor cell is adopaminergic neuron progenitor cell for the treatment of the Parkinson'sdisease.
 62. The production method according to claim 60, wherein thedopaminergic neuron progenitor cell is a dopaminergic neuronproliferative progenitor cell.
 63. A polynucleotide selected from thefollowing (i), (ii), (iii) and (iv): (i) a polynucleotide comprising thenucleotide sequence of SEQ ID NO: 1; (ii) a polynucleotide encoding aprotein which consists of an amino acid sequence encoded by a nucleotidesequence of SEQ ID NO: 1 in which one or more nucleotides are inserted,substituted and/or deleted, and/or one or more nucleotides are added toone or both of ends, and which is functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; (iii) apolynucleotide which hybridizes under stringent conditions to apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1,and which encodes a protein functionally equivalent to a proteinconsisting of the amino acid sequence of SEQ ID NO: 2; and (iv) apolynucleotide which has 70% or more identity with a polynucleotideconsisting of the nucleotide sequence of SEQ ID NO: 1, and which encodesa protein functionally equivalent to a protein consisting of the aminoacid sequence of SEQ ID NO:
 2. 64. The polynucleotide according to claim63, which is derived from a human, a mouse, a rat, a bovine, a dog or achimpanzee.
 65. The polynucleotide according to claim 63, which isselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 25 and SEQ ID NO:
 27. 66. A protein selected fromthe following (v), (vi), (vii) and (viii): (v) a protein comprising theamino acid sequence of SEQ ID NO: 2; (vi) a protein which consists of anamino acid sequence of SEQ ID NO: 2 in which one or more amino acids areinserted, substituted and/or deleted, and/or one or more amino acids areadded to one or both of ends, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; (vii) aprotein which is encoded by a polynucleotide which hybridizes understringent conditions to a polynucleotide which encodes the amino acidsequence of SEQ ID NO: 2, and which is functionally equivalent to aprotein consisting of the amino acid sequence of SEQ ID NO: 2; and(viii) a protein which consists of an amino acid sequence having 70% ormore identity with the amino acid sequence of SEQ ID NO: 2, and which isfunctionally equivalent to a protein consisting of the amino acidsequence of SEQ ID NO:
 2. 67. The protein according to claim 66, whichis a type I transmembrane protein.
 68. A protein comprising apolypeptide consisting of at least 5 amino acid residues or all of anamino acid sequence of amino acids 248 to 397 or 792 to 877 of SEQ IDNO: 2, or a polypeptide consisting of at least 5 amino acid residues orall of an amino acid sequence of amino acids 28 to 927 of SEQ ID NO: 2,amino acids 16 to 1267 of SEQ ID NO: 4, amino acids 1 to 550 of SEQ IDNO: 6, amino acids 1 to 542 of SEQ ID NO: 8, amino acids 1 to 418 of SEQID NO: 10, amino acids 76 to 964 of SEQ ID NO: 12, amino acids 40 to 928of SEQ ID NO: 15, amino acids 1 to 540 of SEQ ID NO: 17, amino acids 40to 1106 of SEQ ID NO: 19, amino acids 24 to 1524 of SEQ ID NO: 22, aminoacids 43 to 1018 of SEQ ID NO: 24, amino acids 43 to 908 of SEQ ID NO:26 or amino acids 1 to 866 of SEQ ID NO:
 28. 69. A polynucleotideencoding a protein according to claim
 68. 70. Use of a protein accordingto claim 66 as an index for detecting or selecting a dopaminergic neuronprogenitor cell.
 71. The use according to claim 70, wherein thedopaminergic neuron progenitor cell is a dopaminergic neuronproliferative progenitor cell.